Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof

ABSTRACT

A recombinant micro-organism producing resveratrol by a pathway in which phenylalanine ammonia lyase (PAL) produces trans-cinnamic acid from phenylalanine, cinnamate 4-hydroxylase (C4H) produces 4-coumaric acid from said trans-cinnamic acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA, or in which L-phenylalanine- or tyrosine-ammonia lyase (PAL/TAL) produces 4-coumaric acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA. The micro-organism may be a yeast, fungus or bacterium including  Saccharomyces cerevisiae, E. coli, Lactococcus lactis, Aspergillus niger , or  Aspergillus oryzae.

This application is a continuation in part of application Ser. No.11/816,847 filed Aug. 22, 2007 as the national stage ofPCT/EP2006/060154, filed Feb. 21, 2006.

FIELD OF THE INVENTION

This invention relates generally to the production of the polyphenolresveratrol or an oligomeric or glycosidically bound derivative thereofsuch as its β-glucoside piceid using microbial cells. Furthermore, itrelates to the use of naturally occurring or recombinant micro-organismsthat produce resveratrol or such a derivative for production of food,feed and beverages.

BACKGROUND OF THE INVENTION

Production of chemicals from micro-organisms has been an importantapplication of biotechnology. Typically, the steps in developing such abio-production method may include 1) selection of a propermicro-organism host, 2) elimination of metabolic pathways leading toby-products, 3) deregulation of desired pathways at both enzyme activitylevel and the transcriptional level, and 4) overexpression ofappropriate enzymes in the desired pathways. In preferred aspect, thepresent invention has employed combinations of the steps above toredirect carbon flow from phenylalanine or tyrosine through enzymes ofthe plant phenylpropanoid pathway which supplies the necessary precursorfor the desired biosynthesis of resveratrol.

Resveratrol (or 3,4,5-trihydroxystilbene) is a phytophenol belonging tothe group of stilbene phytoalexins, which are low-molecular-masssecondary metabolites that constitute the active defense mechanism inplants in response to infections or other stress-related events.Stilbene phytoalexins contain the stilbene skeleton(trans-1,2-diphenylethylene) as their common basic structure: that maybe supplemented by addition of other groups as well (Hart and Shrimpton,1979, Hart, 1981). Stilbenes have been found in certain trees(angio-sperms, gymnosperms), but also in some herbaceous plants (inspecies of the Myrtaceae, Vitaceae and Leguminosae families). Saidcompounds are toxic to pests, especially to fungi, bacteria and insects.Only few plants have the ability to synthesize stilbenes, or to producethem in an amount that provides them sufficient resistance to pests.

The synthesis of the basic stilbene skeleton is pursued by stilbenesynthases. So far, two enzymes have been designated as a stilbenesynthase; pinosylvine synthase and resveratrol synthase. To date, thegroundnut (Arachis hypogaea) resveratrol synthase has been characterisedin most detail, such that most of the properties are known (Schoppnerand Kindl, 1984). Substrates that are used by stilbene synthases aremalonyl-CoA, cinnamoyl-CoA or coumaroyl-CoA. These substances occur inevery plant because they are used in the biosynthesis of other importantplant constituents as well such as flavonoids, flower pigments andlipids. Resveratrol (FIG. 1 trans-form) consists of two closelyconnected phenol rings and belongs therefore to the polyphenols. Whilepresent in other plants, such as eucalyptus, spruce, and lily, and inother foods such as mulberries and peanuts, resveratrol's most abundantnatural sources are Vitis vinifera, -labrusca, and -muscadine(rotundifolia) grapes, which are used to make wines. The compound occursin the vines, roots, seeds, and stalks, but its highest concentration isin the skin (Celotti et al., 1996), which contains 50-100 μg/g. (Jang etal. 1997). During red wine vinification the grape skins are included inthe must, in contrast to white wine vinification, and thereforeresveratrol is found in small quantities in red wine only. Resveratrolhas, besides its antifungal properties, been recognized for itscardioprotective- and cancer chemopreventive activities; it acts as aphytoestrogen, an inhibitor of platelet aggregation (Kopp et al, 1998;Gehm et al 1997; Lobo et al 1995), and an antioxidant (Jang et al.,1997; Huang 1997). These properties explain the so-called FrenchParadox, i.e. the wine-drinking French have a low incidence of coronaryheart disease despite a low-exercise, high-fat diet. Recently it hasbeen shown that resveratrol can also activate the SIR2 gene in yeast andthe analogous human gene SIRT1, which both play a key role in extendinglife span. Ever since, attention is very much focused on the life-spanextending properties of resveratrol (Hall, 2003, Couzin, 2004).

American health associations, such as the Life Extension Foundation, arepromoting the vast beneficial effects of this drug, and therebypropelling the ideal conditions for a successful commercialisation.Present production processes rely mostly upon extraction of resveratrol,either from the skin of grape berries, or from Knot weed. This is alabour intensive process and generates low yield which, therefore,prompts an incentive for the development of novel, more efficient andhigh-yielding production processes.

In plants, the phenylpropanoid pathway is responsible for the synthesisof a wide variety of secondary metabolic compounds, including lignins,salicylates, coumarins, hydroxycinnamic amides, pigments, flavonoids andphytoalexins. Indeed formation of resveratrol in plants proceeds throughthe phenylpropanoid pathway. The amino acid L-phenylalanine is convertedinto trans-cinnamic acid through the non-oxidative deamination byL-phenylalanine ammonia lyase (PAL) (FIG. 2). Next, trans-cinnamic acidis hydroxylated at the para-position to 4-coumaric acid(4-hydroxycinnamic acid) by cinnamate-4-hydroxylase (C4H), a cytochromeP450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450reductase (CPR). The 4-coumaric acid, is subsequently activated to4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). Finally,resveratrol synthase (VST) catalyses the condensation of a phenylpropaneunit of 4-coumaroyl-CoA with malonyl CoA, resulting in formation ofresveratrol.

Recently, a yeast was disclosed that could produce resveratrol from4-coumaric acid that is found in small quantities in grape must (Beckeret al. 2003). The production of 4-coumaroyl-CoA, and concomitantresveratrol, in laboratory strains of S. cerevisiae, was achieved byco-expressing a heterologous coenzyme-A ligase gene, from hybrid poplar,together with the grapevine resveratrol synthase gene (vst1). The othersubstrate for resveratrol synthase, malonyl-CoA, is already endogenouslyproduced in yeast and is involved in de novo fatty-acid biosynthesis.The study showed that cells of S. cerevisiae could produce minuteamounts of resveratrol, either in the free form or in theglucoside-bound form, when cultured in synthetic media that wassupplemented with 4-coumaric acid.

However, said yeast would not be suitable for a commercial applicationbecause it suffers from low resveratrol yield, and requires addition of4-coumaric acid, which is only present in few industrial media. In orderto facilitate and broaden the application of resveratrol as both apharmaceutical and neutraceutical, it is therefore highly desirable toobtain a yeast that can produce resveratrol directly from glucose,without addition of 4-coumaric acid.

A recent study (Ro and Douglas, 2004) describes the reconstitution ofthe entry point of the phenylpropanoid pathway in S. cerevisiae byintroducing PAL, C4H and CPR from Poplar. The purpose was to evaluatewhether multienzyme complexes (MECs) containing PAL and C4H arefunctionally important at this entry point into phenylpropanoidmetabolism. By feeding the recombinant yeast with [3H]-phenylalanine itwas found that the majority of metabolized [3H]-phenylalanine wasincorporated into 4-[3H]-coumaric acid, and that phenylalaninemetabolism was highly reduced by inhibiting C4H activity. Moreover,PAL-alone expressers metabolized very little phenylalanine into cinnamicacid. When feeding [3H]-phenylalanine and [14C]-trans-cinnamic acidsimultaneously to the triple expressers, no evidence was found forchanneling of the endogenously synthesized [3H]-trans-cinnamic acid into4-coumaric acid. Therefore, efficient carbon flux from phenylalanine to4-coumaric acid via reactions catalyzed by PAL and C4H does not appearto require channeling through a MEC in yeast, and sheer biochemicalcoupling of PAL and C4H seems to be sufficient to drive carbon flux intothe phenylpropanoid pathway. In yet another study (Hwang et al., 2003)production of plant-specific flavanones by Escherichia coli was achievedthrough expression of an artificial gene cluster that contained threegenes of a phenyl propanoid pathway of various heterologous origins; PALfrom the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomycescoelicolor, and chalcone synthase (CHS) from the licorice plantGlycyrrhiza echinata. These pathways bypassed C4H, because the bacterial4CL enzyme ligated coenzyme A to both trans-cinnamic acid and 4-coumaricacid. In addition, the PAL from Rhodotorula rubra uses bothphenylalanine and tyrosine as the substrates. Therefore, E. coli cellscontaining the gene clusters and grown on glucose, produced smallamounts of two flavanones, pinocembrin (0.29 g/l) from phenylalanine andnaringenin (0.17 g/l) from tyrosine. In addition, large amounts of theirprecursors, 4-coumaric acid and trans-cinnamic acid (0.47 and 1.23mg/liter respectively), were accumulated. Moreover, the yields of thesecompounds could be increased by addition of phenylalanine and tyrosine.

Whereas the enzyme from dicotylic plants utilizes only phenylalanineefficiently, several studies indicated that PAL from monocotylic plants,and some micro-organisms, utilizes tyrosine as well (Rösier et al.,1997). In such reactions the enzyme activity is designated tyrosineammonia lyase (TAL, FIG. 3). Conversion of tyrosine by TAL results inthe direct formation of 4-coumaric acid without the intermediacy of C4Hand CPR. Mostly both activities reside on the same polypeptide and havevery similar catalytic efficiencies, in spite of large differences in Kmand turnover number. However, most PAL/TAL enzymes from plants preferphenylalanine rather than tyrosine. The level of TAL activity is mostlylower than PAL activity, but the magnitude of this difference variesover a wide range. For example, the parsley enzyme has a Km forphenylalanine of 15-25 μM and for tyrosine 2.0-8.0 mM with turnovernumbers 22 s⁻¹ and 0.3 s⁻¹ respectively. In contrast, the maize enzymehas a Km for phenylalanine only 15-fold higher than for tyrosine, andturnover numbers about 10-fold higher. Moreover, in the red yeasts,Rhodotorula glutinis (Rhodosporidium toruloides) and -rubra, the TALcatalytic activity is close to the PAL catalytic activity with a ratioof TAL/PAL of approximately 0.58. It is believed that the PAL enzyme inthese yeasts degrades phenylalanine as a catabolic function and thetrans-cinnamic acid formed is converted to benzoate and other cellularmaterials, whereas in plants it is thought to be merely a regulatoryenzyme in the biosynthesis of lignin, isoflavonoids and otherphenylpropanoids.

Recently, an open reading frame was found in the bacterium Rhodobactercapsulatus that encodes a hypothetical biosynthetic tyrosine ammonialyase (TAL) that is involved in the biosynthesis of the chromophore ofthe photoactive yellow protein (Kyndt et al., 2002). This was the firsttime that a PAL-homologous gene was found in bacteria. The TAL gene wasisolated and overproduced in Escherichia coli. The Km and kcat valuesfor the conversion of tyrosine to 4-coumaric acid were 15.6 μM and 27.7s⁻¹ respectively, and for conversion of L-phenylalanine totrans-cinnamic acid were 1277 μM and 15.1 s⁻¹ respectively. As aconsequence of the smaller Km and a slightly larger kcat, the enzymeshows a strong preference for tyrosine over L-phenylalanine, with acatalytic efficiency (Km/kcat) for tyrosine of approximately 150-foldlarger than for phenylalanine. The kinetic studies established thattyrosine, and not L-phenylalanine, is the natural substrate of theenzyme under physiological conditions. Very recently a study describedthe heterologous coexpression of phenylalanine ammonia lyase,cinnamate-4-hydroxylase, 4-coumarate-Coa-ligase and chalcone synthase,for the production of flavonoids in E. coli (Watts et al., 2004). Thesimultaneous expression of all four genes, however, was not successfulbecause of a nonfunctional cinnamate-4-hydroxylase. The substitution ofphenylalanine ammonia lyase and cinnamate-4-hydroxylase by a newtyrosine ammonia lyase that was cloned from Rhodobacter sphaeroides,could, however, solved the problem and led to high-level production ofthe flavonone naringenin. Furthermore, said tyrosine ammonia lyase fromRhodobacter sphaeroides is also used for heterologous production of4-coumaric acid (i.e. para-hydroxycinnamic acid) in Escherichia coli(US-A-2004059103). Evenmore, further methods for development of abiocatalyst for conversion of glucose into 4-coumaric acid aredescribed. US-A-2004023357 discloses a tyrosine ammonia lyase from theyeast Trichosporon cutaneum for the production of coumaric acid inEscherichia coli and Saccharomyces cerevisiae. US-A-2001053847 describesthe incorporation of the wild type PAL from the yeast Rhodotorulaglutinis into E. coli, underlining the ability of the wildtype PAL toconvert tyrosine directly to 4-coumaric acid. Moreover, there is alsoexemplification of incorporation of the wildtype PAL from the yeastRhodotorula glutinis, plus a plant C4H and CPR into E. coli and S.cerevisiae. Also described is the development of a biocatalyst throughmutagenesis of the wild type yeast PAL Rhodotorula glutinis withenhanced TAL activity (US-A-6521748). Neither of the aforementionedpatents claim the incorporation of 4CL and VST for the production ofresveratrol.

Recently, evidence was shown that the filamentous fungi A. oryzaecontained the enzyme chalcone synthase (CHS) that is normally involvedin the biosynthesis of flavonoids, such as naringenin, in plants(Seshime et al., 2005). Indeed it was also shown that A. oryzaecontained the major set of genes responsible forphenylpropanoid-flavonoid metabolism, i.e PAL, C4H and 4CL. However,there is no evidence that A. oryzae contained a stilbene synthase suchas resveratrol synthase.

The present invention now provides a micro-organism having an operativemetabolic pathway comprising at least one enzyme activity, said pathwayproducing 4-coumaric acid and producing resveratrol therefrom or anoligomeric or glycosidically-bound derivative thereof. Such amicro-organism may be naturally occurring and may be isolated bysuitable screening procedures, but more preferably is geneticallyengineered.

Preferably, said resveratrol or derivative is produced in a reactioncatalysed by an enzyme in which endogenous malonyl-CoA is a substrate,and preferably said resveratrol is produced from 4-coumaroyl-CoA.

Said resveratrol or derivative is preferably produced from4-coumaroyl-CoA by a resveratrol synthase which is preferably expressedin said micro-organism from nucleic acid coding for said enzyme which isnot native to the micro-organism.

Generally herein, unless the context implies otherwise, references toresveratrol include reference to oligomeric or glycosidically boundderivatives thereof, including particularly piceid.

Thus, in certain preferred embodiments, said resveratrol synthase is aresveratrol synthase (EC 2.3.1.95) from a plant belonging to the genusof Arachis, e.g. A. glabatra, A. hypogaea, a plant belonging to thegenus of Rheum, e.g. R. tataricum, a plant belonging to the genus ofVitus, e.g. V. labrusca, V. riparaia, V. vinifera, or any one of thegenera Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus, Cissus,Calochortus, Polygonum, Gnetum, Artocarpus, Nothofagus, Phoenix,Festuca, Carex, Veratrum, Bauhinia or Pterolobium.

Preferably, said 4-coumaric acid is produced from trans-cinnamic acid,suitably by an enzyme in a reaction catalysed by said enzyme in whichoxygen is a substrate, NADH or NADPH is a cofactor and NAD⁺ or NADP⁺ isa product.

Thus, said 4-coumaric acid may be produced from trans-cinnamic acid by acinnamate 4-hydroxylase, which preferably is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

In certain preferred embodiments, including those referred to in theparagraphs above, said cinnamate-4-hydroxylase is acinnamate-4-hydroxylase (EC 1.14.13.11) from a plant or amicro-organism. The plant may belong to the genus of Arabidopsis, e.g.A. thaliana, a plant belonging to the genus of Citrus, e.g. C. sinensis,C. x paradisi, a plant belonging to the genus of Phaseolus, e.g. P.vulgaris, a plant belonging to the genus of Pinus, e.g. P. taeda, aplant belonging to the genus of Populus, e.g. P. deltoides, P.tremuloides, P. trichocarpa, a plant belonging to the genus of Solanum,e.g. S. tuberosum, a plant belonging to the genus of Vitus, e.g. Vitusvinifera, a plant belonging to the genus of Zea, e.g. Z. mays, or otherplant genera e.g. Ammi, Avicennia, Camellia, Camptotheca, Catharanthus,Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta,Saccharum, Vigna. The micro-organism might be a fungus belonging to thegenus Aspergillus, e.g. A. oryzae.

Preferably, said 4-coumaric acid is produced from tyrosine in a reactioncatalysed by an enzyme in which ammonia is produced and suitably, said4-coumaric acid is produced from tyrosine by a L-phenylalanine ammonialyase or a tyrosine ammonia lyase, e.g. tyrosine ammonia lyase (EC4.3.1.5) from yeast or bacteria. Suitably, the tyrosine ammonia lyase isfrom the yeast Rhodotorula rubra or from the bacterium Rhodobactercapsulatus.

Optionally, said tyrosine ammonia lyase is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

Alternatively, said trans-cinnamic acid may be produced fromL-phenylalanine in a reaction catalysed by an enzyme in which ammonia isproduced and suitably said trans-cinnamic acid is formed fromL-phenylalanine by a phenylalanine ammonia lyase.

In certain preferred embodiments, said L-phenylalanine ammonia lyase isa L-phenylalanine ammonia lyase (EC 4.3.1.5) from a plant or amicro-organism. The plant may belong to the genus of Arabidopsis, e.g.A. thaliana, a plant belonging to the genus of Brassica, e.g. B. napus,B. rapa, a plant belonging to the genus of Citrus, e.g. C. reticulata,C. clementinus, C. limon, a plant belonging to the genus of Phaseolus,e.g. P. coccineus, P. vulgaris, a plant belonging to the genus of Pinus,e.g. P. banksiana, P. monticola, P. pinaster, P. sylvestris, P. taeda, aplant belonging to the genus of Populus, e.g. P. balsamifera, P.deltoides, P. Canadensis, P. kitakamiensis, P. tremuloides, a plantbelonging to the genus of Solanum, e.g. S. tuberosum, a plant belongingto the genus of Prunus, e.g. P. avium, P. persica, a plant belonging tothe genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genusof Zea, e.g. Z. mays or other plant genera e.g. Agastache, Ananas,Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia,Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita,Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus,Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca,Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago,Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea, Petroselinum,Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Quercus,Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria,Stylosanthes, Triticum, Trifolium, Triticum, Vaccinium, Vigna, Zinnia.The micro-organism might be a fungus belonging to the genus Agaricus,e.g. A. bisporus, a fungus belonging to the genus Aspergillus, e.g. A.oryzae, A. nidulans, A. fumigatus, a fungus belonging to the genusUstilago, e.g. U. maydis, a bacterium belonging to the genusRhodobacter, e.g. R. capsulatus, a yeast belonging to the genusRhodotorula, e.g. R. rubra.

Suitably, said L-phenylalanine ammonia lyase is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

Preferably, 4-coumaroyl-CoA is formed in a reaction catalysed by anenzyme in which ATP and CoA are substrates and ADP is a product andsuitably 4-coumaroyl-CoA is formed in a reaction catalysed by a4-coumarate-CoA ligase.

Said 4-coumarate-CoA ligase may be a 4-coumarate-CoA ligase (EC6.2.1.12) from a plant, a micro-organism or a nematode. The plant maybelong to the genus of Abies, e.g. A. beshanzuensis, B. firma, B.holophylla, a plant belonging to the genus of Arabidopsis, e.g. A.thaliana, a plant belonging to the genus of Brassica, e.g. B. napus, B.rapa, B. oleracea, a plant belonging to the genus of Citrus, e.g. C.sinensis, a plant belonging to the genus of Larix, e.g. L. decidua, L.gmelinii, L. griffithiana, L. himalaica, L. kaempferi, L. laricina, L.mastersiana, L. occidentalis, L. potaninii, L. sibirica, L. speciosa, aplant belonging to the genus of Phaseolus, e.g. P. acutifolius, P.coccineus, a plant belonging to the genus of Pinus, e.g. P. armandii P.banksiana, P. pinaster, a plant belonging to the genus of Populus, e.g.P. balsamifera, P. tomentosa, P. tremuloides, a plant belonging to thegenus of Solanum, e.g. S. tuberosum, a plant belonging to the genus ofVitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g.Z. mays, or other plant genera e.g. Agastache, Amorpha, Cathaya, Cedrus,Crocus, Pestuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium,Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana,Nothotsuga, Oryza, Pelargonium, Petroselinum, Rhyscomitrella, Picea,Prunus, Pseudolarix, Pseudotsuga, Rosa, Rubus, Ryza, Saccharum, Suaeda,Thellungiella, Triticum, Tsuga. The micro-organism might be afilamentous fungi belonging to the genus Aspergillus, e.g. A. flavus, A.nidulans, A. oryzae, A. fumigatus, a filamentous fungus belonging to thegenus Neurospora, e.g. N. crassa, a fungus belonging to the genusYarrowia, e.g. Y. lipolytica, a fungus belonging to the genus ofMycosphaerella, e.g. M. graminicola, a bacterium belonging to the genusof Mycobacterium, e.g. M. bovis, M. leprae, M. tuberculosis, a bacteriumbelonging to the genus of Neisseria, e.g. N. meningitidis, a bacteriumbelonging to the genus of Streptomyces, e.g. S. coelicolor, a bacteriumbelonging to the genus of Rhodobacter, e.g. R. capsulatus, a nematodebelonging to the genus Ancylostoma, e.g. A. ceylanicum, a nematodebelonging to the genus Caenorhabditis, e.g. C. elegans, a nematodebelonging to the genus Haemonchus, e.g. H. contortus, a nematodebelonging to the genus Lumbricus, e.g. L. rubellus, a nematode belongingto the genus Meloidogyne, e.g. M. hapla, a nematode belonging to thegenus Strongyloidus, e.g. S. rattii, S. stercoralis, a nematodebelonging to the genus Pristionchus, e.g. P. pacificus.

Optionally, a NADPH:cytochrome P450 reductase (CPR) has beenrecombinantly introduced into said micro-organism. This may be a plantCPR introduced into a non-plant micro-organism. Alternatively, a nativeNADPH:cytochrome P450 reductase (CPR) has been overexpressed in saidmicro-organism.

In certain preferred embodiments, including those referred to in theparagraphs above, said NADPH:cytochrome P450 reductase is aNADPH:cytochrome P450 reductase (EC 1.6.2.4) from a plant belonging tothe genus of Arabidopsis, e.g. A. thaliana, a plant belonging to thegenus of Citrus, e.g. C. sinensis, C. x paradisi, a plant belonging tothe genus of Phaseolus, e.g. P. vulgaris, a plant belonging to the genusof Pinus, e.g. P. taeda, a plant belonging to the genus of Populus, e.g.P. deltoides, P. tremuloides, P. trichocarpa, a plant belonging to thegenus of Solanum, e.g. S. tuberosum, a plant belonging to the genus ofVitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g.Z. mays, or other plant genera e.g. Ammi, Avicennia, Camellia,Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum,Physcomitrella, Ruta, Saccharum, Vigna.

Whilst the micro-organism may be naturally occurring, preferably atleast one copy of at least one genetic sequence encoding a respectiveenzyme in said metabolic pathway has been recombinantly introduced intosaid micro-organism.

Additionally or alternatively to introducing coding sequences coding fora said enzyme, one may provide one or more expression signals, such aspromoter sequences, not natively associated with said coding sequence insaid organism. Thus, optionally, at least one copy of a genetic sequenceencoding a tyrosine ammonia lyase is operatively linked to an expressionsignal not natively associated with said genetic sequence in saidorganism, and/or at least one copy of a genetic sequence encoding aL-phenylalanine ammonia lyase is operatively linked to an expressionsignal not natively associated with said genetic sequence in saidorganism.

Optionally, at least one copy of a genetic sequence encoding cinnamate4-hydroxylase, whether native or not, is operatively linked to anexpression signal not natively associated with said genetic sequence insaid organism.

Optionally, at least one copy of a genetic sequence encoding a4-coumarate-CoA ligase, whether native or not, is operatively linked toan expression signal not natively associated with said genetic sequencein said organism.

Optionally, at least one copy of a genetic sequence encoding aresveratrol synthase, whether native or not, is operatively linked to anexpression signal not natively associated with said genetic sequence insaid organism.

Expression signals include nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Such sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

In certain aspects the invention provides a metabolically engineeredmicro-organism having an operative metabolic pathway in which a firstmetabolite is transformed into a second metabolite in a reactioncatalysed by a first enzyme, said reaction step producing ammonia, andin which said second metabolite is transformed into a third metabolitein a reaction catalysed by a second enzyme, in which oxygen is asubstrate, NADPH or NADH is a cofactor and NADP⁺ or NAD⁺ is a product,and in which said third metabolite is transformed into a fourthmetabolite in a reaction catalysed by a third enzyme in which ATP andCoA is a substrate, and ADP is a product, and in which said fourthmetabolite is transformed into a fifth metabolite in a reactioncatalysed by a fourth enzyme in which endogenous malonyl-CoA is asubstrate.

The present invention also provides a metabolically engineeredmicro-organism having an operative metabolic pathway in which a firstmetabolite is transformed into a said third metabolite catalysed by afirst enzyme, said reaction step producing ammonia, without theinvolvement of said second enzyme, and in which said third metabolite istransformed into a said fourth metabolite in a reaction catalysed by asaid third enzyme in which ATP and CoA is a substrate, and ADP is aproduct, and in which said fourth metabolite is transformed into a saidfifth metabolite in a reaction catalysed by a said fourth enzyme inwhich endogenous malonyl-CoA is a substrate.

The micro-organisms described above include ones containing one or morecopies of an heterologous DNA sequence encoding phenylalanine ammonialyase operatively associated with an expression signal, and containingone or more copies of an heterologous DNA sequence encodingcinnamate-4-hydroxylase operatively associated with an expressionsignal, and containing one or more copies of an heterologous DNAsequence encoding 4-coumarate-CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal.

They include also ones lacking cinnamate-4-hydroxylase activity, andcontaining one or more copies of a heterologous DNA sequence encodingtyrosine ammonia lyase operatively associated with an expression signal,and containing one or more copies of an heterologous DNA sequenceencoding 4-coumarate-CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal.

In the present context the term “micro-organism” relates to microscopicorganisms, including bacteria, microscopic fungi, including yeast.

More specifically, the micro-organism may be a fungus, and morespecifically a filamentous fungus belonging to the genus of Aspergillus,e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging tothe genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus,S. exiguus, S. sevazzi, S. uvarum, a yeast belonging to the genusKluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K.thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilisC. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeastbelonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P.sorbitophila, or other yeast genera, e.g. Cryptococcus, Debaromyces,Hansenula, Pichia, Yarrowia, Zygosaccharomyces or Schizosaccharomyces.Concerning other micro-organisms a non-exhaustive list of suitablefilamentous fungi is supplied: a species belonging to the genusPenicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor,Mortierella, Trichoderma.

Concerning bacteria a non-exhaustive list of suitable bacteria is givenas follows: a species belonging to the genus Bacillus, a speciesbelonging to the genus Escherichia, a species belonging to the genusLactobacillus, a species belonging to the genus Lactococcus, a speciesbelonging to the genus Corynebacterium, a species belonging to the genusAcetobacter, a species belonging to the genus Acinetobacter, a speciesbelonging to the genus Pseudomonas, etc.

The preferred micro-organisms of the invention may be S. cerevisiae, A.niger, A. oryzae, E. coli, L. lactis or B. subtilis.

The constructed and engineered micro-organism can be cultivated usingcommonly known processes, including chemostat, batch, fed-batchcultivations, etc.

Thus, the invention includes a method for producing resveratrol or anoligomeric or glycosidically-bound derivative thereof comprisingcontacting a non-plant cell with a carbon substrate in the substantialabsence of an external source of 4-coumaric acid, said cell having thecapacity to produce resveratrol or an oligomeric or glycosidically-boundderivative thereof under the conditions, in which the micro-organism maybe selected from the group consisting of fungi and bacteria, especiallyyeast.

Said carbon substrate is optionally selected from the group offermentable carbon substrates consisting of monosaccharides,oligosaccharides and polysaccharides, e.g. glucose, fructose, galactose,xylose, arabinose, mannose, sucrose, lactose, erythrose, threose, and/orribose. Said carbon substrate may additionally or alternatively beselected from the group of non-fermentable carbon substrates includingethanol, acetate, glycerol, and/or lactate. Said non-fermentable carbonsubstrate may additionally or alternatively be selected from the groupof amino acids and may be phenylalanine and/or tyrosine.

In an alternative aspect, the invention includes a method for producingresveratrol or an oligomeric or glycosidically-bound derivative thereofthrough heterologous expression of nucleotide sequences encodingphenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate-CoAligase and resveratrol synthase and also a method for producingresveratrol through heterologous expression of nucleotide sequencesencoding tyrosine ammonia lyase, 4-coumarate-CoA ligase and resveratrolsynthase.

The invention as described above has allowed the production of yeastcells producing high levels of resveratrol. Accordingly, the inventionincludes a micro-organism composition comprising micro-organism cellsand at least 0.4 μg/g resveratrol on a dry weight basis produced in saidmicro-organism cells, preferably comprising at least 0.5 μg/g of saidresveratrol, more preferably at least 200 μg/g. The stated level ofresveratrol can be found in the yeast cells themselves. The compositionmay essentially consist of said yeast cells.

The resveratrol producing microorganisms described above and thepinosylvin producing organisms described in WO2008/009728 coulddesirably be improved to produce higher yields by redirecting the fluxthrough the metabolism of the microorganism.

One option is to increase the amount malonyl-CoA available for furtherconversion into pinosylvin and resveratrol or other stilbenoids.Increasing the amount of malonoyl-CoA will have a positive effect on theproduction of all stilbenes of the type given in formula I

R1, R2, R3, R4, and R5 independently are either —H or —OH becausemalonyl-CoA is responsible for the upper ring. The stilbene that isproduced depends on the other organic acid component involved, wherecinnamic acid gives pinosylvin and coumaric acid gives resveratrol.Caffeic acid will give piceatannol.

By increasing the amount of available malonyl-CoA the yield ofstilbenoid can be increased. A first method involves overexpression ofACC1 to create the increased supply.

Thus, the invention further includes a recombinant micro-organism havingan operative metabolic pathway in which one or more stilbenes accordingto the general formula I:

are formed from a precursor optionally hydroxy-substitutedphenyl-2-propenoic acid or ester thereof and malonyl-CoA by the actionof a stilbene synthase, wherein the amount of malonyl-CoA available foruse in said pathway has been increased by providing more than a nativeexpression level of an enzyme catalysing the reaction

ATP+acetyl-CoA+HCO3−=ADP+phosphate−+malonyl-CoA.

Preferably, said more than native expression level of said enzyme hasbeen provided by replacing a native promoter of a gene expressing saidenzyme with a promoter providing a higher level of expression. Forinstance, said native promoter is replaced with a strong constitutiveyeast promoter. The strong constitutive promoter may be the promoter ofone of the yeast genes TDH3, ADH1, TPI1, ACT1, GPD, TEF1, TEF2, and PGI.The promoter may optionally be native to the yeast in which stilbenoidproduction is to be produced.

Alternatively or additionally, said more than native expression level ofsaid enzyme has been provided by recombinantly introducing into saidmicro-organism at least one exogenous genetic sequence encoding a saidenzyme. This may be an acetyl coenzymeA carboxylase (ACC1—EC No.6.4.1.2).

A second and independent strategy which also increases the yield of thestilbenes is overexpression of CPR.

Thus, the micro-organism may be recombinantly engineered to produce morethan a native amount of a cytochrome P450 reductase (CPR). This may beby replacing a native promoter of a gene expressing said CPR with apromoter providing a higher level of expression, for instance with astrong constitutive yeast promoter such as the promoter of one of theyeast genes TDH3, ADH1, TPI1, ACT1 GPD, TEF1, TEF2, and PGI, whichoptionally may be native to the yeast itself.

The micro-organism may comprise recombinantly introduced genesexpressing a phenylalanine ammonia lyase, a cinnamate 4-hydroxylaseand/or a coumarate-CoA ligase or appropriate enzymes for otherstilbenes.

Effect of Overexpressing ACC1

Acetyl CoenzymeA carboxylase (ACC1 EC-Number 6.4.1.2) generatesmalonyl-CoA according to the below reaction:

(ACC1 Reaction)

ATP+acetyl-CoA+HCO3−=ADP+phosphate−+malonyl-CoA

By overexpressing ACC1 more malonyl-CoA is built up and this extra poolof malonyl-CoA is expected to generate more stilbenoids since thestilbene synthase reaction requires malonyl-CoA as building block forstilbene synthesis according to the reactions below:

[Resveratrol Synthase Reaction EC-Number 2.3.1.95]

3 malonyl-CoA+4-coumaroyl-CoA=4 CoA+3,4′,5-trihydroxystilbene+4 CO2

[Stilbene Synthase Reaction]

3 malonyl-CoA+cinnamoyl-CoA=4 CoA+3,5-dihydroxystilbene+4 CO2

[General for any Hydroxyl Stilbene Synthase]

3 malonyl-CoA+hydroxycinnamoyl-CoA=4 CoA+hydroxystilbene+4 CO2

Other appropriate organic acids substituting for 4-coumaroyl-CoA produceother stilbenoids.

Effect of Overexpressing CPR

Hydroxylases, such as cinnamate 4-hyroxylase (1.14.13.11), arecytochrome P450 monooxygenases that catalyse the insertion of one atomof oxygen into an organic substrate while the other oxygen atom isreduced to water. This reaction requires NADPH according to the belowreaction for the hydroxylation of cinnamic acid:

trans-cinnamic acid+NADPH+H⁺+O₂=4-hydroxycinnamic acid+NADP⁺+H₂O

The active site of cytochrome P450 hydroxylases contains a heme ironcenter. The iron is tethered to the protein via a thiolate ligandderived from a cysteine residue. In general the mechanism is as follows:

1. The resting state of the protein is as oxidized Fe3+.2. Binding of the substrate, cinnamic acid, initiates electron transportand oxygen binding.3. Electrons are supplied to the p450 hydroxylase by another protein,either cytochrome P450 reductase (CPR), ferredoxins, or cytochrome b5 toreduce the heme iron.4. Molecular oxygen is bound and split by the now reduced iron.5. An iron-bound oxidant, oxidizes the substrate to an alcohol or anepoxide, regenerating the resting state of the p450 hydroxylase.

As described above CPR act as an electron carrier and donor for theNADPH dependent cytochrome P450 hydroxylase reaction. Thus byoverexpressing CPR more electrons (NADPH) are generated for the NADPHdependent hydroxylation leading to more coumaric acid, and as aconsequence more coumaric acid leads to more resveratrol by theresveratrol pathway. Similar considerations apply in the production ofother stilbenoids.

Resveratrol or an oligomeric or glycosidically-bound derivative thereofor other stilbenoids so produced may be used as a nutraceutical in adairy product or a beverage such as beer.

Resveratrol produced according to the invention may be cis-resveratrolor trans-resveratrol, but it is to be expected that the trans- form willnormally predominate, as with other stilbenoids.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist in the ready understanding of the above description of theinvention reference has been made to the accompanying drawings in which:

FIG. 1 shows the chemical structure of trans-resveratrol;

FIG. 2 shows the phenylpropanoid pathway utilising phenylalanine ammonialyase acting on L-phenylalanine; and

FIG. 3 shows the alternative pathway utilising phenylalanine ammonialyase acting on L-tyrosine.

FIG. 4 shows the HPLC-chromatograms of extracts of S. cerevisiae strainsFSSC-PALC4H4CLVST, FSSC-TAL4CLVST, grown on 100 g/l galactose. Achromatogram of 60 nanogram of pure resveratrol is included.

FIG. 5 shows the UV absorption spectrum for pure trans-resveratrol andtrans-resveratrol produced by S. cerevisiae strain FSSC-PALC4H4CLVST,grown on 100 g/l galactose.

FIG. 6 shows the HPLC-chromatograms of extracts from E. coli strainsFSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose.

FIG. 7 shows the HPLC-chromatograms of extracts from E. coli strainsFSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose with additionof 20 mg/l coumaric acid. The UV absorption spectrum fortrans-resveratrol produced in strain FSEC-TAL4CLVST is included.

The invention will be further described and illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Isolation of Genes Encoding PAL, TAL, C4H, CPR, 4CL,and VST

Phenylalanine ammonia lyase (PAL2) (Cochrane et al., 2004; SEQ ID NO: 1,2), cinnamate 4-hydroxylase (C4H) (Mizutani et al., 1997; SEQ ID NO: 3,4) and 4-coumarate:CoenzymeA ligase (4CL1) (Hamberger and Hahlbrock2004; Ehlting et al., 1999; SEQ ID NO: 5, 6) were isolated via PCR fromA. thaliana cDNA (BioCat, Heidelberg, Germany) using the primers intable 1. PAL2 and 4CL1 were chosen amongst several A. thalianahomologues due to favourable kinetic parameters towards cinnamic acidand coumaroyl-CoA, respectively (Cochrane et al., 2004; Hamberger andHahlbrock 2004; Ehlting et al., 1999).

The coding sequence of resveratrol synthase (VST) from Rheum tataricum(Samappito et al., 2003; SEQ ID NO: 7, 8) and tyrosine ammonia lyase(TAL) from Rhodobacter capsulatus (Kyndt et al., 2002; SEQ ID NO: 11,12) were codon optimized for expression in S. cerevisiae using theonline service backtranslation tool at www.entelechon.com, yieldingsequence SEQ ID NO: 9, 10 and SEQ ID NO: 13, 14 respectively. Oligos forthe synthetic gene assembly were constructed at MWG Biotech and thesynthetic gene was assembled by PCR using a slightly modified methodprotocol of from Martin et al. (2003) described below.

TABLE 1 Primers and restriction sites for the amplification of genesPrimer for amplification of gene* Restriction Restriction(Restriction sites are underlined) Gene site: primer site: vector5′-CGGAATTCTCATGGATCAAATCGAAGCAATGTT PAL2 EcoRI EcoRI5′-CGACTAGTTTAGCAAATCGGAATCGGAGC PAL2 SpeI SpeI5′-CGCTCGAGAT ATGGACCTCCTCTTGCTGGA C4H XhoI XhoI5′-CGGGTACCTTAACAGTTCCTTGGTTTCATAAC C4H KpnI KpnI5′-GCTCTAGACCT ATGGCGCCACAAGAACAAGCAGTTT  4CL1 XbaI SpeI5′-GCGGATCCCCT TCACAATCCATTTGCTAGTTT TGCC 4CL1 BamHI BglII5′-CC GGATCCAAATGGCCCCAGAAGAGAGCAGG VST BamHI BamIII5′-CG CTCGAGTTAAGTGATCAATGGAACCGAAGACAG VST XhoI XhoI5′-CCGAATTCCCATGACCCTGCAATCTCAAACAGCTAAAG TAL EcoRI EcoRI5′-CCACTAGTTTAAGCAGGTGGATCGGCAGCT TAL SpeI SpeI5′-CCCTCGAGATCATGCCGTTTGGAATAGACAACACCGA  CPR1 XhoI XhoI5′-CCAAGCTTATCGGGCTGATTACCAGACATCTTCTTG CPR1 HindIII HindIII5′-CCGGATCCCCATGTCCTCTTCTTCTTCTTCGTCAAC AR2 BamhI BamhI5′-CCCTCGAGGTGAGTGTGTGGCTTCAATAGTTT CG AR2 XhoI XhoI *SEQ ID NOs 29-32

Primers from MWG for the assembly of the synthetic gene were dissolvedin milliQ-water to a concentration of 100 pmole/μl. An aliquot of 5 μlof each primer was combined in a totalmix and then diluted 10-fold withmilliQ water. The gene was assembled via PCR using 5 μl diluted totalmixper 50 μl as template for fusion DNA polymerase (Finnzymes). The PCRprogramme was as follows: Initial 98° C. for 30 s., and then 30 cycleswith 98° C. for 10 s., 40° C. for 1 min. and 72° C. at 1 min./1000basepairs, and a final 72° C. for 5 min. From the resulting PCRreaction, 20 μl was purified on 1% agarose gel. The result was a PCRsmear and the regions around the wanted size were cut out from agarosegel and purified using the QiaQuick Gel Extraction Kit (Qiagen). A finalPCR with the outer primers (for TAL and VST) in table 1 rendered therequired TAL and VST genes. Point mutations were corrected using eitherthe Quickchange site directed mutagenesis II kit (Stratagene, La Jolla,Calif.), or using PCR from overlapping error free DNA stretches fromseveral different E. coli subclones.

NADPH:Cytochrome P450 reductase (CPR) from A. thaliana (AR2) (Mizutaniand Ohta, 1998; SEQ ID NO: 17, 18) and from S. cerevisiae (CPR1) (Aoyamaet al., 1978; SEQ ID NO: 15, 16), were isolated from A. thaliana cDNA(BioCat, Heidelberg, Germany) and S. cerevisae genomic DNA,respectively, using the primers in table 1.

Example 2 Construction of a Yeast Vector for Expression of PAL

The gene encoding PAL, isolated as described in example 1, wasreamplified by PCR using forward- and reverse primers, with 5′ overhangscontaining EcoR1 and Spe1 restriction sites (table 1). The amplified PALPCR product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1digested pESC-URA vector (Stratagene), resulting in vector pESC-URA-PAL.The sequence of the gene was verified by sequencing of two differentclones.

Example 3 Construction of a Yeast Vector for Expression of PAL and C4H

The gene encoding C4H, isolated as described in example 1, was amplifiedby PCR using the forward- and reverse primers, with 5′ overhangscontaining Xho1 and Kpn1 restriction sites. The amplified C4HPCR-product was digested with Xho1/Kpn1 and ligated into similarlydigested pESC-URA-PAL vector. The resulting plasmid, pESC-URA-PAL-C4H,contained the genes encoding PAL and C4H under the control of thedivergent GAL1/GAL10 promoter. The sequence of the gene encoding C4H wasverified by sequencing of two different clones.

Example 4 Construction of a Yeast Vector for Expression of 4CL

The gene encoding 4CL was isolated as described in example 1. Theamplified 4CL PCR-product was digested with Xba1/BamH1 and ligated intoSpe1/BglII digested pESC-TRP vector (Stratagene), resulting in vectorpESC-TRP-4CL. Two different clones of pESC-TRP-4CL were sequenced toverify the sequence of the cloned gene.

Example 5 Construction of a Yeast Vector for Expression of 4CL and VST

The gene encoding VST was isolated as described in example 1. Theamplified synthetic VST gene was digested with BamH1/Xho1 and ligatedinto BamH1/Xho1 digested pESC-TRP-4CL (example 4). The resultingplasmid, pESC-TRP-4CL-VST, contained the genes encoding 4CL and VSTunder the control of the divergent GAL1/GAL10 promoter. The sequence ofthe gene encoding VST was verified by sequencing of two different clonesof pESC-TRP-4CL-VST.

Example 6 Construction of a Yeast Vector for Expression of TAL

The gene encoding TAL was isolated as described in example 1. Theamplified synthetic TAL gene was digested with EcoR1/Spe1 and ligatedinto EcoR1/Spe1-digested pESC-URA vector. The resulting plasmid,pESC-URA-TAL, contained the gene encoding for TAL under the control ofthe divergent GAL1/GAL10 promoter. The sequence was verified bysequencing of two different clones of pESC-URA-TAL.

Example 7 Construction of a Yeast Vector for Overexpression of S.cerevisiae Endogenous CPR

The gene encoding CPR from S. cerevisiae (CPR1) was isolated asdescribed in example 1. The amplified CPR1 gene was digested withXho1/HindIII and ligated into Xho1/HindIII-digested pESC-LEU vector(Stratagene), resulting in vector pESC-LEU-CPR1. The sequence wasverified by sequencing of two different clones of pESC-LEU-CPR1.

Example 8 Construction of a Yeast Vector for Overexpression of A.thaliana CPR (AR2)

The gene encoding CPR from A. thaliana (AR2) was isolated as describedin example 1. The amplified AR2 gene was digested with BamH1/Xho1 andligated into BamH1/Xho1 digested pESC-LEU vector (Stratagene), resultingin vector pESC-LEU-AR2.

The sequence was verified by sequencing of two different clones ofpESC-LEU-AR2.

Example 9 Expression of the Pathway to Resveratrol in the Yeast S.cerevisiae Using PAL, C4H, 4CL and VST

Yeast strains containing the appropriate genetic markers weretransformed with the vectors described in examples 2, 3, 4, 5, 6, 7 and8, separately or in combination. The transformation of the yeast cellwas conducted in accordance with methods known in the art, for instance,by using competent cells or by electroporation (see, e.g., Sambrook etal., 1989). Transformants were selected on medium lacking uracil and/ortryptophan and streak purified on the same medium.

S. cerevisiae strain CEN.PK 113-5D (MATa ura3) was transformedseparately with the vector pESC-URA-PAL (example 2), yielding the strainFSSC-PAL, and with pESC-URA-PAL-C4H (example 3), resulting in the strainFSSC-PALC4H. S. cerevisiae strain FS01267 (MATa trp1 ura3) wasco-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL (example 4), andthe transformed strain was named FSSC-PALC4H4CL. The same strain wasalso co-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL-VST (example5), resulting in the strain FSSC-PALC4H4CLVST.

Example 10 Expression of the Pathway to Resveratrol in S. cerevisiaeUsing TAL, 4CL and VST

S. cerevisiae strain CEN.PK 113-5D (MATa ura3) was transformedseparately with the vector pESC-URA-TAL (example 6), yielding the strainFSSC-TAL. S. cerevisiae strain FS01267 (MATa trp1 ura3) wasco-transformed with pESC-URA-TAL (example 6) and pESC-TRP-4CL (example4), and the transformed strain was named FSSC-TAL4CL. The same strainwas also co-transformed with pESC-URA-TAL and pESC-TRP-4CL-VST (example5), resulting in the strain FSSC-TAL4CLVST. Transformants were selectedon medium lacking uracil and or tryptophan and streak purified on thesame medium.

Example 11 Expression of the Pathway to Resveratrol in S. Cerevisiaewith Overexpressed Endogenous CPR

S. cerevisiae strain FS01277 (MATa ura3 leu2 trp1) was co-transformedwith vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), andpESC-LEU-CPR1 (example 7). The transformed strain was namedFSSC-PALC4H4CLVSTCPR. Transformants were selected on medium lackinguracil and/or tryptophan and streak purified on the same medium.

Example 12 Expression of the Pathway to Resveratrol in S. cerevisiaewith Overexpressed A. thaliana CPR (AR2)

S. cerevisiae strain FS01277 (MATa ura3 leu2 trp1) was co-transformedwith vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), andpESC-LEU-AR2 (example 8). The transformed strain was namedFSSC-PALC4H4CLVSTAR2. Transformants were selected on medium lackinguracil and or tryptophan and streak purified on the same medium.

Example 13 Fermentation with Recombinant Yeast Strains in Shake Flasks

The recombinant yeast strains were inoculated from agar plates with asterile inoculation loop and grown in 200 ml defined mineral medium(Verduyn et al, 1992) that contained vitamins, trace elements, 5 g/lglucose and 40 g/l or 100 g/l galactose. The 500 ml stoppered shakeflasks were incubated for three days at 30° C. and 160 rpm.

Example 14 Extraction of Resveratrol

Cells were harvested by centrifugation 5000 g for 5 minutes. An aliquotof 50 ml of supernatant was extracted once with 20 ml ethyl acetate. Theethyl acetate was freeze dried and the dry product redissolved in 0.7 mlmethanol and filtered into HPLC vials.

The cell pellet from 200 ml medium was dissolved in 1 to 2 ml water anddivided into 3 fastprep tubes and broken with glass beads. The crudeextracts from the three tubes were pooled into 10 ml 100% methanol in a50 ml sartorius tube and extracted on a rotary chamber for 48 hours in adark cold room at 4° C. After 48 hours the cell debris was removed viacentrifugation for 5 min. at 5000 g and the methanol was removed byfreeze-drying overnight. The dried residue was redissolved in 1 mlphosphate-citrate buffer pH 5.4 and 10 units beta-glucosidase fromalmonds was added (Sigma) to release resveratrol from putativelyglucoside-bound forms. The mixture was incubated for three hours at 37°C. and then extracted twice with 1 ml ethyl acetate. The combined ethylacetate was freeze dried and the dry residue was redissolved in 0.7 mlmethanol and filtered into HPLC vials.

Example 15 Analysis of Resveratrol Thin Layer Chromatography

A method based upon thin layer chromatography that enabled the quickseparation of cinnamic, coumaric and resveratrol on the same TLC-platewas developed for quick screening analysis. An aliquot of 1 ml culturecontaining both cells and supernatant were extracted with 500 microliterethyl acetate and centrifuged for 30 s. at 13000 rpm with amicrocentrifuge. The ethyl acetate was dried and redissolved inmethanol. The extracts were analyzed on Silica G plates (0.2 mm AlugramSIL G/UV₂₅₄, Macherey-Nagel) containing a fluorescent indicator. Themobile phase was a mixture of chloroform, ethyl acetate and formic acid(25:10:1).

HPLC

For quantitative analysis of cinnamic acid, coumaric acid, andresveratrol, samples were subjected to separation by high-performanceliquid chromatography (HPLC) Agilent Series 1100 system (HewlettPackard) prior to uv-diode-array detection at λ=306 nm. A Phenomenex(Torrance, Calif., USA) Luna 3 micrometer C18 (100×2.00 mm) column wasused at 40° C. As mobile phase a gradient of acetonitrile and milliqwater (both containing 50 ppm trifluoroacetic acid) was used at a flowof 0.4 ml/min. The gradient profile was linear from 15% acetonitrile to100% acetonitrile over 20 min. The elution times were approximately 3.4min. for coumaric acid, 5.5 min. for free trans-resveratrol and 6.8 min.for cinnamic acid.

Pure resveratrol standard was purchased from Cayman chemical company,whereas pure coumaric acid and cinnamic acid standards were purchasedfrom and Sigma.

Results

Strains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST, were cultivated on 100 g/lgalactose as described in example 13, and analyzed for their content ofintracellular resveratrol according to example 14 and 15. Additionally,a control strain FSSC-control was included that contained the emptyvectors pESC-URA and pESC-TRP only. The HPLC-analysis showed thatstrains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST contained a component witha retention time of 5.5 min. that was identical to trans-resveratrol(FIG. 4). Said result was confirmed by the UV absorption spectra thatwere similar to the absorption spectrum of pure trans-resveratrol (FIG.5) as well, with a λ_(max) of approximately 306 nm.

The results, therefore, demonstrated the presence of an activephenyl-propanoid pathway in S. cerevisiae that led to in vivo productionof trans-resveratrol. The production of resveratrol can most likely beimproved by cultivating the strains under well-defined growth conditionsin batch- and continuous cultures, and/or optimizing theexpression/activities of the individual enzymes.

Example 16 Construction of a Bacterial Vector for Expression of TAL inEscherichia coli

The gene encoding TAL, isolated as described in Example 1, wasreamplified by PCR from the plasmid pESC-URA-TAL (example 6) using theforward primer 5′-CCG CTCGAG CGG ATG ACC CTG CAA TCT CAA ACA GCT AAAG-3′ SEQ ID NO 33 and the reverse primer 5′-GC GGATCC TTA AGC AGG TGGATC GGC AGC T-3′ SEQ ID NO 34 with 5′ overhangs containing therestriction sites XhoI and BamHI, respectively. The introduction ofrestriction sites at the 5′ and 3′ ends of the gene allowed ligation ofthe restricted PCR product into a pET16b vector (Novagen), digested withXhoI and BamHI to yield pET16b-TAL. The pET16b vector contained both theampicillin resistance gene, and the T7 promoter. Hence, above procedureresulted in a vector with an antibiotic selection marker that containedthe gene encoding for TAL under the control of the T7 promoter. Thesequence of the gene encoding TAL was verified by sequencing of oneclone of pET16b-TAL.

Example 17 Construction of a Bacterial Vector for Expression of 4CL andVST in Escherichia coli

The gene encoding VST, isolated as described in example 1, was cut outwith the restriction enzymes BamHI and XhoI from the digested plasmidpESC-TRP-4CL-VST (example 5), which contains the genes encoding 4CL andVST. The VST gene was ligated into a pET26b vector (Novagen), containingthe kanamycin resistance gene, digested with BamHI and SalI to yieldpET26b-VST. The restriction enzymes XhoI and SalI have compatible ends,which enabled proper ligation. The pET26b vector contained both thekanamycin resistance gene, and the T7 promoter. Hence, above procedureresulted in a vector with an antibiotic selection marker that containedthe gene encoding for VST under the control of the T7 promoter. The geneencoding for 4CL, isolated as described in example 1, was reamplified byPCR from the plasmid pESC-URA-4CL-VST (example 5) using the forwardprimer 5′-TG CCATGG CA ATGGCGCCAC AAGAACAAGC AGTTT-3′ SEQ ID NO 35 andthe reverse primer 5′-GC GGATCC CCT TCA CAA TCC ATT TGC TAG TTT TGCC-3′SEQ ID NO 36 with 5′ overhangs containing the restriction sites NcoI andBamHI, respectively. The introduction of restriction sites at the 5′ and3′ ends of the gene allowed ligation of the restricted PCR product intoa pET16b vector (Novagen) digested with NcoI and BamHI. The resultingplasmid, pET16b-4CL, contained the gene encoding for 4CL under thecontrol of the T7 promoter. Both the T7 promoter and the gene encodingfor 4CL were reamplified as one fragment by PCR from the plasmidpET16b-4CL using the forward primer 5′-TT GCGGCCGC AAA TCT CGA TCC CGCGAA ATT AAT ACG-3′ SEQ ID NO 37 and the reverse primer 5′-CG CTCGAG CCTTCA CAA TCC ATT TGC TAG TTT TGCC-3′ SEQ ID NO 38 with 5′ overhangs,containing the restriction sites NotI and XhoI, respectively. Theintroduction of restriction sites at the 5′ and 3′ ends of the DNAfragment allowed ligation of the restricted PCR product into the plasmidpET26b-VST that was digested with NotI and XhoI before ligation. Theresulting plasmid, pET26b-VST-4CL, contained the two genes 4CL and VSTthat each were under control of an individual T7 promoter.

Example 18 Expression of the Pathway to Resveratrol in Escherichia Coli,Using TAL, 4CL and VST

The transformation of the bacterial cell was conducted in accordancewith methods known in the art, for instance, by using competent cells orby electroporation (see, e.g., Sambrook et al., 1989). The E. colistrain BL21 (DE3) (Novagen) was co-transformed with the two vectorspET16b-TAL (example 16) and pET26b-VST-4CL (Example 17), resulting instrain FSEC-TAL4CLVST. In addition, E. coli strain BL21 (DE3) wasco-transformed with the two empty vectors pET16b (Novagen) and pET26b(Novagen), resulting in strain FSEC-control, which was used as a controlstrain. Transformants were selected on Luria-Bertani (LB) medium with100 μg/ml ampicillin and 60 μg/ml kanamycin.

Example 19 Fermentation with Recombinant Escherichia coli Strains inShake Flasks

Pre-cultures of Escherichia coli BL21 (DE3) were grown in glass tubes at160 rpm and 37° C. in 7 ml of LB medium containing 100 μg/ml ampicillinand 60 μg/ml kanamycin. Exponentially growing precultures were used forinoculation of 500 ml baffled shake flasks that contained 200 ml LBmedium supplemented with 50 g/l glucose, 5 g/l K₂HPO₄, 80 μg/mlampicilin and 50 μg/ml kanamycin, which were incubated at 160 rpm and37° C. After 5 hours, isopropyl β-thiogalactopyranoside (IPTG) was addedat a final concentration of 1 mM, as an inducer of the T7 promoter thatwas in front of each of the three genes TAL, 4CL and VST. After anincubation period of 48 hours at 37° C., the cells were harvested andsubjected to extraction procedures and analysed for the presence ofproduced resveratrol.

Example 20 Extraction and Analysis of Resveratrol in Escherichia coli

Extraction and analysis was performed using the methods as described inexample 14 and 15.

Results

Strain FSEC-TAL4CLVST and FSEC-control, were cultivated on 50 g/lglucose as described in example 19, and analyzed for their content ofintracellular resveratrol according to example 14 and 15. TheHPLC-analysis showed that strain FSEC-TAL4CLVST did contain considerableamounts of a component with a retention time of 3.4 min., which isidentical to coumaric acid (FIG. 6). However, the extract did notcontain a component that eluted at the same time as trans-resveratrol.Said result, therefore, indicated that the tyrosine ammonia lyase (TAL)was active indeed, but did not lead to production of detectable amountsof resveratrol. The lack of resveratrol formation, however, could be theresult of; i) a non-functional coumarate-CoA ligase (4CL); ii) anon-functional resveratrol synthase (VST); iii) too low levels ofcoumaric acid, caused by either non-optimal cultivation conditions, ornon-optimal expression/activity of TAL, or branching of coumaric acidinto other products. To evaluate said hypotheses, the strains were grownon similar media as described in example 19 but now in the presence of20 mg/l of coumaric acid. The subsequent HPLC-analysis of extracts ofFSEC-TAL4CLVST indeed showed a cluster of peaks around the sameretention time as trans-resveratrol, which was not observed in extractsof FS-control (FIG. 6). Indeed, the UV absorption spectrum of the peakwith a retention time of 5.5 min. was similar to the spectrum of puretrans-resveratrol (FIG. 7), whereas no such spectrum could be obtainedfor peaks in the control strain. The results, therefore, stronglysuggest the presence of an active phenylpropanoid pathway in Escherichiacoli, which can lead to production of resveratrol. Most likely theproduction of resveratrol without addition of coumaric acid can beachieved by cultivating the strains under well-defined growth conditionsin batch- and continuous cultures, and/or optimizing theexpression/activities of the individual enzymes.

Example 21 Construction of a Bacterial Vector for Expression of PAL andC4H in Lactococcus lactis

The plasmid pSH71 and derivatives thereof, which is used in thefollowing examples, is a bifunctional shuttle vector with multipleorigins of replication from Escherichia coli and Lactococcus lactis.With that, the host range specificity traverses Escherichia coli andother species of lactic acid bacteria. Though transformations inLactococcus lactis usually proceed without problems, putative difficulttransformations in other species of lactic acid bacteria can, therefore,be overcome by using Escherichia coli as an intermediate host for theconstruction of recombinant plasmids. The plasmid contains one or moremarker genes to allow the microorganism that harbour them to be selectedfrom those which do not. The selection system that is used forLactococcus lactis is based upon dominant markers, e.g. resistanceagainst erythromycin and chloramphenicol, but systems based upon genesinvolved in carbohydrate metabolism, peptidases and food grade markers,have also been described. In addition, the plasmid contains promoter-and terminator sequences that allow the expression of the recombinantgenes. Suitable promoters are taken from genes of Lactococcus lactise.g. lacA. Furthermore, the plasmid contains suitable unique restrictionsites to facilitate the cloning of DNA fragments and subsequentidentification of recombinants.

In the examples below the plasmid contains either the erythromycineresistance gene, designated as pSH71-ERY^(r), or the chloramphenicolresistance gene, designated as pSH71-CM^(r)

The gene encoding PAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-ERY^(r) vector that contains the lacA promoterfrom Lactococcus lactis. The resulting plasmid, pSH71-ERY^(r)-PAL,contains the gene encoding PAL under the control of the lacA promoterfrom Lactococcus lactis. The gene encoding C4H, isolated as described inexample 1, is reamplified by PCR from the plasmid pESC-URA-PAL-C4H(example 3) using forward- and reverse primers, with 5′ overhangscontaining suitable restriction sites. The introduction of saidrestriction sites at the 5′ and 3′ ends of the gene allows ligation ofthe restricted PCR product into a digested pSH71-CM^(r) vector to yieldpSH71-CM^(r)-C4H. The lacA promoter and the gene encoding C4H arereamplified as one fragment by PCR from the plasmid pSH71-CM^(r)-C4Husing forward- and reverse primers, with 5′ overhangs containingsuitable restriction sites. The introduction of said restriction sitesat the 5′ and 3′ ends of the DNA fragment allows ligation of therestricted PCR product into the digested plasmid pSH71-ERY^(r)-PAL. Theresulting plasmid, pSH71-ERY^(r)-PAL-C4H, contains the genes encodingPAL and C4H that are each under the control of an individual lacApromoter. The sequence of the genes encoding PAL and C4H is verified bysequencing of two different clones of pSH71-ERY^(r)-PAL-C4H.

Example 22 Construction of a Bacterial Vector for Expression of TAL inLactococcus lactis

The gene encoding for TAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-TAL (example 6) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-ERY^(r) vector. The resulting plasmid,pSH71-ERY^(r)-TAL, contains the gene encoding for TAL under the controlof the lacA promoter from Lactococcus lactis. The sequence of the geneencoding for TAL is verified by sequencing of two different clones ofpSH71-ERY^(r)-TAL.

Example 23 Construction of a Bacterial Vector for Expression of 4CL andVST in Lactococcus lactis

The gene encoding 4CL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-CM^(r) vector. The resulting plasmid,pSH71-CM^(r)-4CL, contains the gene encoding for 4CL under the controlof the lacA promoter from Lactobacillus lactis. The gene encoding VST,isolated as described in example 1, is reamplified by PCR from theplasmid pESC-TRP-4CL-VST (example 5) using forward- and reverse primers,with 5′ overhangs containing suitable restriction sites. Theintroduction of said restriction sites at the 5′ and 3′ ends of the geneallows ligation of the restricted PCR product into a digestedpSH71-ERY^(r) vector. The resulting plasmid, pSH71-ERY^(r)-VST, containsthe gene encoding VST under the control of the lacA promoter fromLactococcus lactis. The lacA promoter and the gene encoding VST arereamplified as one fragment by PCR from the plasmid pSH71-ERY^(r)-VSTusing forward- and reverse primers, with 5′ overhangs containingsuitable restriction sites. The introduction of said restriction sitesat the 5′ and 3′ ends of the DNA fragment allows ligation of therestricted PCR product into the digested plasmid pSH71-CM^(r)-4CL. Theresulting plasmid, pSH71-CM^(r)-4CL-VST, contains the genes encoding 4CLand VST that are each under the control of their individual lacApromoter.

The sequence of the genes encoding 4CL and VST is verified by sequencingof two different clones of pSH71-CM^(r)-4CL-VST.

Example 24 Expression of the Pathway to Resveratrol in Lactococcuslactis

Lactococcus lactis strains are transformed with the vectors described inexamples 21, 22 and 23, separately or in combination. The transformationof the bacterial cell is conducted in accordance with methods known inthe art, for instance, by using competent cells or by electroporation(see, e.g., Sambrook et al., 1989). Transformants are selected on mediumcontaining the antibiotics erythromycin and chloramphenicol and streakpurified on the same medium.

Lactococcus lactis strain MG1363 is transformed separately with thevector pSH71-ERY^(r)-TAL (example 22), yielding the strain FSLL-TAL;with pSH71-ERY^(r)-PAL-C4H (example 21), yielding the strain FSLL-PALC4Hand with pSH71-CM^(r)-4CL-VST (example 23), yielding strain FSLL-4CLVST.In addition, Lactococcus lactis strain MG1363 is co-transformed withpSH71-ERY^(r)-TAL (example 22) and pSH71-CM^(r)-4CL-VST (example 23),and the transformed strain is named FSLL-TAL4CLVST. The same strain isalso co-transformed with pSH71-ERY^(r)-PAL-C4H (example 21), andpSH71-CM^(r)-4CL-VST (example 23), resulting in the strainFSLL-PALC4H4CLVST.

Example 25 Fermentation with Recombinant Lactococcus lactis Strains inFermentors

The recombinant yeast strains can be grown in fermenters operated asbatch, fed-batch or chemostat cultures.

Batch and Fed-Batch Cultivations

The microorganism is grown in a baffled bioreactor with a working volumeof 1.5 liters under anaerobic, aerobic or microaerobic conditions. Allcultures are incubated at 30° C., at 350 rpm. A constant pH of 6.6 ismaintained by automatic addition of 10 M KOH. Cells are grown on lactosein defined MS10 medium supplemented with the following components toallow growth under aerobic conditions: MnSO₄ (1.25×10⁻⁵ g/l), thiamine(1 mg/l), and DL-6,8-thioctic acid (2.5 mg/l). The lactose concentrationis, for example 50 g/l. The bioreactors are inoculated with cells fromprecultures grown at 30° C. in shake flasks on the medium describedabove buffered with threefold-higher concentrations of K₂HPO₄ andKH₂PO₄. Anaerobic conditions are ensured by flushing the medium with N₂(99.998% pure) prior to inoculation and by maintaining a constant flowof 50 ml/min of N₂ through the headspace of the bioreactor duringcultivation. The bioreactors used for microaerobic and aerobiccultivation are equipped with polarographic oxygen sensors that arecalibrated with air (DOT, 100%) and N₂ (DOT, 0%). Aerobic conditions areobtained by sparging the bioreactor with air at a rate of 1 vvm toensure that the DOT is more than 80%. During microaerobic experimentsthe DOT is kept constant 5% by sparging the reactor with gas composed ofa mixture of N₂ and atmospheric air, at a rate of 0.25 vvm.

Chemostat Cultures

In chemostat cultures the cells can be grown in, for example, 1-Lworking-volume Applikon laboratory fermentors at 30° C. and 350 rpm. Thedilution rate (D) can be set at different values, e.g. at 0.050 h⁻¹,0.10 h⁻¹, 0.15 h⁻¹, or 0.20 h⁻¹. The pH is kept constant, e.g at 6.6, byautomatic addition of 5 M KOH, using the growth medium described above,supplemented with antifoam (50 μl/l). The concentration of lactose canbe set at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/lor 18.0 g/l. The bioreactor is inoculated to an initial biomassconcentration of 1 mg/l and the feed pump is turned on at the end of theexponential growth phase.

An anaerobic steady state is obtained by introducing 50 ml/min of N₂(99.998% pure) into the headspace of the bioreactor. Different anoxicsteady states can obtained by sparging the reactor with 250 ml/min ofgas composed of N₂ (99.998% pure) and atmospheric air at various ratios.The oxygen electrode is calibrated by sparging the bioreactor with air(100% DOT) and with N₂ (0% DOT).

For all conditions, the gas is sterile filtered before being introducedinto the bioreactor. The off gas is led through a condenser cooled tolower than −8° C. and analyzed for its volumetric content of CO₂ and O₂by means of an acoustic gas analyser.

Cultivations are considered to be in steady state after at least 5residence times, and if the concentrations of biomass and fermentationend products remain unchanged (less than 5% relative deviation) over thelast two residence times.

Example 26 Extraction and Analyis of Resveratrol in Lactococcus lactis

Extraction and analysis is performed using the methods as described inexamples 14 and 15.

Example 27 Construction of a Fungal Vector for Expression of PAL and C4Hin Species Belonging to the Genus Aspergillus

The plasmid that is used in the following examples, is derived frompARp1 that contains the AMA1 initiating replication sequence fromAspergillus nidulans, which also sustains autonomous plasmid replicationin A. niger and A. oryzae (Gems et al., 1991). Moreover, the plasmid isa shuttle vector, containing the replication sequence of Escherichiacoli, and the inherent difficult transformations in Aspergillus nigerand Aspergillus oryzae can therefore overcome by using Escherichia colias an intermediate host for the construction of recombinant plasmids.The plasmid contains one or more marker genes to allow the microorganismthat harbour them to be selected from those which do not.

The selection system can be either based upon dominant markers e.g.resistance against hygromycin B, phleomycin and bleomycin, orheterologous markers e.g amino acids and the pyrG gene. In addition theplasmid contains promoter- and terminator sequences that allow theexpression of the recombinant genes. Suitable promoters are taken fromgenes of Aspergillus nidulans e.g. alcA, glaA, amy, niaD, and gpdA.Furthermore, the plasmid contains suitable unique restriction sites tofacilitate the cloning of DNA fragments and subsequent identification ofrecombinants.

The plasmid used in the following examples contains the strongconstitutive gpdA-promoter and auxotropic markers, all originating fromAspergillus nidulans; the plasmid containing the gene methG that isinvolved in methionine biosynthesis, is designated as pAMA1-MET; theplasmid containing the gene hisA that is involved in histidinebiosynthesis, is designated as pAMA1-HIS.

The gene encoding PAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-MET vector that contains the gpdA promoter fromAspergillus nidulans. The resulting plasmid, pAMA1-MET-PAL contains thegene encoding PAL under the control of the gpdA promoter fromAspergillus nidulans.

The gene encoding C4H, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-HIS vector to yield pAMA1-HIS-C4H. The gpdApromoter and the gene encoding C4H are reamplified as one fragment byPCR from the plasmid pAMA1-HIS-C4H using forward- and reverse primers,with 5′ overhangs containing suitable restriction sites. Theintroduction of said restriction sites at the 5′ and 3′ ends of the DNAfragment allows ligation of the restricted PCR product into the digestedplasmid pAMA1-MET-PAL. The resulting plasmid, pAMA1-MET-PAL-C4H,contains the genes encoding PAL and C4H that are each under the controlof an individual pgdA promoter from Aspergillus nidulans. The sequenceof the genes encoding PAL and C4H is verified by sequencing of twodifferent clones of pAMA1-MET-PAL-C4H.

Example 28 Construction of a Fungal Vector for Expression of TAL inSpecies Belonging to the Genus Aspergillus

The gene encoding for TAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-TAL (example 6) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-MET vector. The resulting plasmid, pAMA1-MET-TAL,contains the gene encoding for TAL under the control of the gpdApromoter from Aspergillus nidulans. The sequence of the gene encodingfor TAL is verified by sequencing of two different clones ofpAMA1-MET-TAL.

Example 29 Construction of a Fungal Vector for Expression of 4CL and VSTin Species Belonging to the Genus Aspergillus

The gene encoding 4CL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-HIS vector that contains the gpdA promoter fromAspergillus nidulans. The resulting plasmid, pAMA1-HIS-4CL contains thegene encoding 4CL under the control of the gpdA promoter fromAspergillus nidulans. The gene encoding VST, isolated as described inexample 1, is reamplified by PCR from the plasmid pESC-TRP-4CL-VST(example 5) using forward- and reverse primers, with 5′ overhangscontaining suitable restriction sites. The introduction of saidrestriction sites at the 5′ and 3′ ends of the gene allows ligation ofthe restricted PCR product into a digested pAMA1-MET vector to yieldpAMA1-MET-VST. The gpdA promoter and the gene encoding VST arereamplified as one fragment by PCR from the plasmid pAMA1-MET-VST usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the DNA fragment allows ligation of the restricted PCRproduct into the digested plasmid pAMA1-HIS-4CL. The resulting plasmid,pAMA1-HIS-4CL-VST, contains the genes encoding 4CL and VST that are eachunder the control of an individual pgdA promoter from Aspergillusnidulans. The sequence of the genes encoding 4CL and VST is verified bysequencing of two different clones of pAMA1-HIS-4CL-VST.

Example 30 Expression of the Pathway to Resveratrol in Aspergillus niger

Aspergillus niger strains are transformed with the vectors described inexamples 27, 28 and 29, separately or in combination. The transformationof the fungal cell is conducted in accordance with methods known in theart, for instance, by electroporation or by conjugation (see, e.g.,Sambrook et al., 1989). Transformants are selected on minimal mediumlacking methionine and/or histidine.

A strain of Aspergillus niger that is auxotrophic for histidine andmethionine, for instance, strain FGSC A919 (see http://www.fgsc.net), istransformed separately with the vector pAMA1-MET-TAL (example 28),yielding the strain FSAN-TAL; with pAMA1-MET-PAL-C4H (example 27),yielding the strain FSAN-PALC4H and with pAMA1-HIS-4CL-VST (example 29),yielding strain FSAN-4CLVST. In addition, Aspergillus niger strain FGSCA919 is co-transformed with pAMA1-MET-TAL (example 28) andpAMA1-HIS-4CL-VST (example 29), and the transformed strain is namedFSAN-TAL4CLVST. The same strain is also co-transformed withpAMA1-MET-PAL-C4H (example 27), and pAMA1-HIS-4CL-VST (example 29),resulting in the strain FSAN-PALC4H4CLVST.

Example 31 Expression of the Pathway to Resveratrol in Aspergillusoryzae

A strain of Aspergillus oryzae that contains a native set of genesencoding for PAL, C4H and 4CL (Seshime et al., 2005) and that isauxotrophic for methionine, is transformed with the vector pAMA1-MET-VST(example 29), yielding the strain FSAO-VST. The transformation of thefungal cell is conducted in accordance with methods known in the art,for instance, by electroporation or by conjugation (see, e.g., Sambrooket al., 1989). Transformants are selected on minimal medium lackingmethionine.

Example 32 Fermentation with Recombinant Strains of Aspergillus nigerand Aspergillus oryzae in Fermentors

The recombinant yeast strains can be grown in fermenters operated asbatch, fed-batch or chemostat cultures.

Batch and Fed-Batch Cultivations

The microorganism is grown in a baffled bioreactor with a working volumeof 1.5 liters under aerobic conditions. All cultures are incubated at30° C., at 500 rpm. A constant pH of 6.0 is maintained by automaticaddition of 10 M KOH, and aerobic conditions are obtained by spargingthe bioreactor with air at a rate of 1 vvm to ensure that the DOT ismore than 80%. Cells are grown on glucose in defined medium consistingof the following components to allow growth in batch cultivations: 7.3g/l (NH₄)₂SO₄, 1.5 g/l KH₂PO₄, 1.0 g/l MgSO₄.7H₂O, 1.0 g/l NaCl, 0.1 g/lCaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/l ZnSO₄.7H₂O, 1.3 mg/lCuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/l MnCl₂.4H₂O and 6.9 mg/lFeSO₄.7H₂O. The glucose concentration is, for example, 10- 20-, 30-, 40-or 50 g/l. To allow growth in fed-batch cultivations the medium iscomposed of: 7.3 g/l (NH₄)₂SO₄, 4.0 g/l KH₂PO₄, 1.9 g/l MgSO₄.7H₂O, 1.3g/l NaCl, 0.10 g/l CaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/lZnSO₄.7H₂O, 1.3 mg/l CuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/lMnCl₂.4H₂O and 6.9 mg/l FeSO₄.H₂O in the batch phase. The reactor isthen fed with, for example, 285 g/kg glucose and 42 g/kg (NH₄)₂SO₄.

Free mycelium from a pre-batch is used for inoculating the batch- andfed-batch cultures. A spore concentration of 2.10⁹ spores/l is used forinoculation of the pre-batch culture at pH 2.5. Spores are obtained bypropagation of freeze-dried spores onto 29 g rice to which the followingcomponents are added: 6 ml 15 g/l sucrose, 2.3 g/l (NH₄)₂5 O₄, 1.0 g/lKH₂PO₄, 0.5 g/l MgSO₄.7H₂O, 0.50 g/l NaCl, 14.3 mg/l ZnSO₄.7H₂O, 2.5 mg/CuSO₄.5H₂O, 0.50 mg/l NiCl₂.6H₂O, and 13.8 mg/l FeSO₄.7H₂O. The sporesare propagated at 30° C. for 7-14 days to yield a black layer of sporeson the rice grains and are harvested by adding 100 ml of 0.1% Tween 20in sterile water. For all conditions, the gas is sterile filtered beforebeing introduced into the bioreactor. The off gas is led through acondenser cooled to lower than −8° C. and analyzed for its volumetriccontent of CO₂ and O₂ by means of an acoustic gas analyser.

Chemostat Cultures

In chemostat cultures the cells can be grown in, for example, 1.5-Lworking-volume Biostat B laboratory fermentors at 30° C. and 500 rpm. Aconstant pH of 6.0 is maintained by automatic addition of 10 M KOH, andaerobic conditions are obtained by sparging the bioreactor with air at arate of 1 vvm to ensure that the DOT is more than 80%. The dilution rate(D) can be set at different values, e.g. at 0.050 h⁻¹, 0.10 h⁻¹, 0.15h⁻¹, or 0.20 h⁻¹. The pH is kept constant, e.g at 6.6, by automaticaddition of 10 M KOH, using a minimal growth medium with the followingcomponents: 2.5 g/l (NH₄)₂SO₄, 0.75 g/l KH₂PO₄, 1.0 g/l MgSO₄.7H₂O, 1.0g/l NaCl, 0.1 g/l CaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/lZnSO₄.7H₂O, 1.3 mg/l CuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/lMnCl₂.4H₂O and 6.9 mg/l FeSO₄.7H₂O. The concentration of glucose can beset at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/l or18.0 g/l. The bioreactor is inoculated with free mycelium from apre-batch culture as described above, and the feed pump is turned on atthe end of the exponential growth phase.

For all conditions, the gas is sterile filtered before being introducedinto the bioreactor. The off gas is led through a condenser cooled tolower than −8° C. and analyzed for its volumetric content of CO₂ and O₂by means of an acoustic gas analyser.

Cultivations are considered to be in steady state after at least 5residence times, and if the concentrations of biomass glucose andcomposition of the off-gas remain unchanged (less than 5% relativedeviation) over the last two residence times.

Example 33 Extraction and Analysis of Resveratrol in Aspergillus nigerand Aspergillus oryzae

Extraction and analysis is performed using the methods as described inexamples 14 and 15.

Example 34 Over-Expression of Native Yeast Genes by Gene TargetingMethod

Over-expression of native yeasts genes with constitutive yeast promotersis carried out by means of a promoter-replacement method based on alinear, PCR-generated gene-targeting substrate and using K. lactis URA3as a recyclable marker described previously (Erdeniz et al, 1997). Thismethod includes the generation of an intermediate yeast strain, wherethe Kluyveromyces lactis URA3 marker gene is integrated in combinationwith two copies of the strong constitutive promoter sequence as a directrepeat on each side of the marker gene. The marker gene is then loopedout through recombination mediated by the direct repeat, an event whichis selected for by plating the intermediate strain on medium containing5-fluoroorotic acid (5-FOA), which is toxic to cells expressing the URA3gene. The result is a yeast strain, in which the native promoter hasbeen replaced with the strong constitutive promoter. Integration of theabove described promoter sequence and marker gene is directed to thecorrect location in the genome by means of PCR-generated targetsequences.

The above described gene-targeting substrate can be constructed by meansof multiple rounds of fusion-PCR. However, to avoid introduction ofPCR-generated mutations, it is beneficial to use a bi-partite or even aquadruple gene-targeting substrate (Erdeniz et al, 1997).

Example 35 Over-Expression of Native Yeast Genes by Bi-Partite GeneTargeting Substrate Method

For example to overexpress a gene with the strong ADH1 promoter, thispromoter has been introduced into intermediate working vectors on eitherside of K. lactis URA3, resulting in the vectors pWAD1, pWAD2,(WO/2005/118814). With these vectors as templates, fragments can beamplified that contain (in the 5′ to 3′ direction) 1) the ADH1 coupledto two thirds of K. lactis URA3 towards the 5′ end, using the primersAD-fw and Int3′, and 2) two thirds of K. lactis URA3 towards the 3′ endcoupled to the ADH1, using the primers Int5′ and AD-rv. Target sequencescorresponding to a 300-500 bp sequence upstream of the gene to beoverexpressed and a 300-500 bp starting with ATG of the gene to beoverexpressed, are amplified from genomic yeast DNA using suitableprimers. The reverse primer used for amplification of the upstreamtarget sequence contains a 5′ overhang that allows fusion to fragment 1described above. The forward primer used for amplification of the targetsequence starting with ATG contains a 5′ overhang that allows fusionwith fragment 2 described above. Following fusion by PCR of the upstreamtarget sequence with fragment 1, and fusion by PCR of fragment 2 withthe target sequence starting with ATG, the two linear substrates asshown in FIG. 6 are ready for transformation.

Example 36 Construction of a Strain Overexpressing Native S. cerevisiaeNADP-Cytochrome P450 Reductase

The native promoter of S. cerevisae NADP-cytochrome P450 reductase CPR1gene (encoded by YHR042W) was replaced with the constitutive S.cerevisiae alcohol dehydrogenase ADH1 promoter via chromosomal promoterexchange using the “bi-partite” PCR-based allele replacement method asdescribed in example 34 and 35. Primers A and B were used to generatefragment CPR1-UP (Table 1) via PCR at a melting temperature of 56° C.using S. cerevisiae genomic DNA as template. Primers C and D were thenused to generate fragment CPR1-S via PCR at a melting temperature of 56°C. using S. cerevisiae genomic DNA as template. Fragments AD1 (k1URA 3′end fused to promoter ADH1) and AD2 (promoter ADH1 fused to K1URA5′-end) were generated via PCR using primers AD-fw and Int3′ and Int5'and AD-ry at a melting temperature of 56° C. and 56° C., respectively.Plasmid pWAD1 was used as template for generation of fragment AD1 andplasmid pWAD2 was used for generating fragment AD2. Fragments CPR_UPwere then fused to fragment AD2 using fusion PCR with primers A andInt3′ at a melting temperature of 56° C. resulting in fusion fragment 1(bi-partite substrate 1). A second fusion PCR was used to fuse fragmentsAD1 and CPR-S with Int5' and primer D at a melting temperature of 56° C.resulting in fusion fragment 2 (bi-partite substrate 2).

Fusion fragments 1 and 2 (bi-partite substrates 1 and 2) were purifiedon agarose gel and used for co-transformation of S. cerevisiae strainFS01528 (Mata, ura3 his3) and the transformants were plated on SC-URAplates and incubated for 2-4 days at 30° C. Transformants were streakpurified on SC-ura plates and incubated another 2 days at 30° C. andthen plated onto 5-FOA (5-fluoroorotic acid) plates. After incubationfor 2 days at 30° C. “pop-out” colonies appeared, which were streakpurified on a new 5-FOA-plate and incubated another 2 days at 30° C. andthen finally transferred to a rich medium plate YPD. The resultingcolonies were analyzed for the presence of fragment of size 1700-1800base pairs using yeast colony PCR with primers A and AD-rev and amelting temperature at 55° C. and an elongation time of 1 minute and 45seconds. One of the positive colonies from the colony PCR containing thenew replaced ADH1 promoter in front of the CPR1 gene was namedFSpADH1-CPR (Mata ura3 his3 pADH1-CPR1) strain.

Table 1 Primers and fragments used in the “bi-partite” PCR-based allelereplacement method to exchange native S. cerevisiae CPR1 promoter withS. cerevisiae ADH1 promoter

Primers A 5′-GTATTCTATATCCACGCCTGCAAAC B 5′-AGTACATACAGGGAACGTCCCTACAGGAACGCAAACTTAAGCTAC C 5′-GCATACAATCAACTATCTCATATACAATGCCGTTTGGAATAGACAACACCD 5′-GCTTCCGCATTACAAATAAAGTCTTCAA AD-fw 5′-GGACGTTCCCTGTATGTACTAGGGGGATCGAAGAAATGATGG Int3′5′-GAGCAATGAACCCAATAACGAAATC 3′ Int5′ 5′-CTTGACGTTCGTTCGACTGATGAGC 3′AD-rv 5′-TGTATATGAGATAGTTGATTGTATGC FragmentsCPR-UP generated from primers A and B(CPR1 gene fragmen upstream of start codon (ATG))CPR-S generated from primers C and D(CPR1 gene fragment containing start codon (ATG))AD1 (ADH1 promoter coupled to two thirds of K.lactis URA3 towards the 5′end generated from primers AD-fw and Int3′)AD2 (Two thirds of K.lactis URA3 towards the 3′ end coupled to the ADH1promoter. Generated from primers Int5′ and AD-rv)Fusion fragment 1 (CPR-UP fragment fused to AD2 fragment)Fusion fragment 2 (AD1 fragment fused to CPR-S fragment)

Example 37

Construction of a strain overexpressing native S. cerevisiae ACC1 gene

The yeast gene ACC1, encoding acetyl-CoA carboxylase, was overexpressedwith the strong constitutive yeast TPI1 promoter as described previously(WO 2005/118814). This was done by replacing the native ACC1 promoterwith the TPI1 promoter, using a slightly modified promoter-replacementmethod based on the bipartite gene-targeting method (Example 1 and 2).One part of the bipartite substrate consisted of two thirds (towards the3′ end) of K. lactis URA3, fused to the TPI1 promoter sequence and atarget sequence corresponding to the beginning of ACC1. The second partof the bipartite substrate consisted of a target sequence upstream ofACC1, fused to the TPI1 promoter sequence and two thirds (towards the 5′end) of K. lactis URA3. Following transformation with the bipartitesubstrate and selection on medium lacking uracil, transformants wereobtained in which the native promoter had been knocked out and replacedwith two copies of the TPI1 promoter sequence as a direct repeat oneither side of the K. lactis URA3 marker gene. A second recombinationevent, resulting in looping out of the selection marker, was selectedfor by replating transformants on medium containing 5′-fluoroorotic acid(5-FOA), which is toxic to cells expressing the URA3 gene. This resultedin a strain, in which the native ACC1 promoter had been replaced withthe TPI1 promoter.

In order to construct part 1 of the bipartite substrate, two thirds(towards the 3′ end) of K. lactis ura3 was amplified from the plasmidpWJ716 using the primers 5′ CTTGACGTTCGTTCGACTGATGAGC 3′ and 5′CTGGAATTCGATGATGTAGTTTCTGG 3′ (Table 2). Moreover, the TPI1 promotersequence was amplified from genomic yeast DNA using the primers 5′CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′TTTTTGATTAAAATTAAAAAAACTTTTTAGTTTATGTATGTGTTTTTTG 3′ and a downstreamtargeting sequence, consisting of the beginning of the ACC1 gene (i.e.,the first 553 bp of the gene) was amplified from genomic yeast DNA usingthe primers 5′ AGTTTTTTTAATTTTAATCAAAAAATGAGCGAAGAAAGCTTATTCGAGTC 3′ and5′ CACCTAAAGACCTCATGGCGTTACC 3′. These three fragments were fused toeach other in two rounds of PCR. First, the TPI1 promoter sequence wasfused to the downstream targeting sequence, using the primers 5′CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′CACCTAAAGACCTCATGGCGTTACC 3′. The resulting product was then fused tothe fragment containing two thirds (towards the 3′ end) of K. lactisURA3. The resulting fragment, 3′ 2/3 K. lactis URA3-pTPI1-DOWN (ACC1)was part 1 of the bipartite gene targeting substrate.

In order to construct part 2 of the bipartite substrate, two thirds(towards the 5′-end) of K. lactis URA3 was amplified from the plasmidpWJ716 using the primers 5′ CGGTCTGCATTGGATGGTGGTAAC 3′ and 5′GAGCAATGAACCCAATAACGAAATC 3′ (Table 2). The TPI1 promoter sequence wasamplified from genomic yeast DNA using the primers 5′CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 3′, and a target sequenceupstream of ACC1 was amplified from genomic DNA using primers 5′TGTTCTGCTCTCTTCAATTTTCCTTTC 3′ and 5′CTGGAATTCGATGATGTAGTTTCTAATTTTCTGCGCTGTTTCG 3′. These three fragmentswere fused in two rounds of PCR. First, the upstream targeting sequencewas fused to the TPI1 promoter sequence, using the primers 5′TGTTCTGCTCTCTTCAATTTTCCTTTC 3′ and 5′CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 3′. The resulting fragmentwas then fused to the fragment containing two thirds (towards the 5′end) of K. lactis URA3, resulting in the fragment UP (ACC1)-pTPI1-5′ 2/3K. lactis URA3, which constituted part 2 of the bipartite gene targetingsubstrate.

Yeast strain FS01372 (MATa ura3 trp1 PADH1-FAS1 pADH1-FAS2) wastransformed with the linear substrates UP (ACC1)-pTPI1-5′ 2/3 K. lactisURA3 and 3′ 2/3 K. lactis URA3-pTPI1-DOWN (ACC1). Transformants wereselected and streak-purified on medium lacking uracil and were thentransferred to plates containing 5-FOA. Pop-out recombinants werestreak-purified on 5-FOA-containing medium. The resulting strain wasnamed FS01392 and had the genotype MATa ura3 trp1 pTPI1-ACC1 PADH1-FAS1pADH1-FAS2). The correct integration of the TPI1 promoter was checked bycolony PCR.

Table 2 Primers and fragments used in the “bi-partite” PCR-based allelereplacement method to exchange native S. cerevisiae ACC1 promoter withthe strong constitutive S. cerevisiae TPI1 promoter

Primers A 5′-CTTGACGTTCGTTCGACTGATGAGC B 5′-CTGGAATTCGATGATGTAGTTTCTGGC 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC D 5′-TTTTTGATTAAAATTAAAAAAACTTTTTAGTTTATGTATGTGTTTTTTG E 5′-AGTTTTTTTAATTTTAATCAAAAAATGAGCGAAGAAAGCTTATTCGAGTCF 5′-CACCTAAAGACCTCATGGCGTTACCG 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC (G = C)H 5′-CACCTAAAGACCTCATGGCGTTACC (H = F) I 5′-CGGTCTGCATTGGATGGTGGTAACJ 5′-GAGCAATGAACCCAATAACGAAATCK 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTCL 5′-CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTGM 5′-TGTTCTGCTCTCTTCAATTTTCCTTTCN 5′-CTGGAATTCGATGATGTAGTTTCTAATTTTCTGCGCTGTTTCGO 5′-TGTTCTGCTCTCTTCAATTTTCCTTTC (O = M)P 5′-CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG (P = L) FragmentsKlactisURA3 sequence generated with primers  A and BTPI1 promoter sequence generated with primers C and DACC1 downstream sequence generated with   primers E and FpTPI1-Down(ACC1) fusion generated from primers  G and HKlactisURA3-pTPI1-DOWN(ACC1) = part 1 of the bipartite substrate generated from primers  A and HKlactisURA3 sequence generated with primers  I and JTPI1 promoter sequence generated with primers  K and LACC1 upstream sequence generated with primers  M and NUP(ACC1)-pTPI1 fusion generated from primers  O and PUP(ACC1)-pTPI1-5′KlactisURA3 = part 2 of the bipartite substrate generated with primers M and J

Example 38 Deletion of Native Yeast Genes by Gene Targeting Method

Gene deletions were performed by a similar method as for geneoverexpressions (Example 1) by means of homologous recombination usingPCR-generated targeting substrates and the K. lactis URA3 gene as aselectable marker, essentially as described in Erdeniz, N., Mortensen,U. H., Rothstein, R. (1997) Genome Res. 7:1174-83. Information on primerdesign for fusion PCR can be found in the same publication. Generally,fusion of DNA fragments was made possible by using primers withappropriately designed 5′ overhangs for amplification of the originalDNA fragments. In all cases, PCR-generated fragments were excised from a1% agarose gel and purified before proceeding with fusion PCR.

Transformants were generally selected on -URA plates, and pop-out of theK. lactis URA3 marker gene was selected for by plating on 5-FOA medium(5-fluoroorotic acid, 750 mg/l). Correct gene deletions were verified byPCR, using primers on both sides of the deleted gene. Generally,PCR-verification of gene deletions was performed by means of colony-PCR.For colony-PCR, a small amount of cells was dispersed in 10 μl H₂O andwas placed at −80° C. for approximately 30 min, followed by 15 min.incubation at 37° C. The cell suspension was then used as template forPCR.

Example 39 Generation of Strain with Deleted Isocitrate Dehydrogenase,IDH1 The Native Yeast Gene IDH1

SEQ ID NO: 59 ATGCTTAACAGAACAATTGCTAAGAGAACTTTAGCCACTGCCGCTCAGGCGGAACGCACCCTACCCAAGAAGTATGGCGGTCGTTTCACCGTCACTTTGATACCTGGTGACGGTGTTGGGAAAGAAATCACTGATTCAGTGAGAACCATTTTTGAGGCTGAAAATATCCCGATCGACTGGGAAACTATAAACATTAAGCAAACAGATCATAAGGAAGGCGTCTATGAAGCTGTTGAGTCTCTAAAGAGAAATAAGATTGGTCTTAAGGGGCTATGGCACACTCCTGCTGACCAAACAGGTCACGGTTCACTAAACGTTGCTTTGCGTAAACAACTAGATATCTACGCCAATGTGGCCCTTTTCAAATCCTTGAAGGGTGTCAAGACTAGAATTCCAGACATAGATTTGATTGTCATTAGAGAAAACACGGAGGGTGAGTTCTCAGGCCTGGAACATGAATCCGTCCCTGGTGTAGTGGAATCTTTGAAAGTTATGACTAGACCTAAGACAGAAAGGATCGCCAGATTTGCCTTTGACTTCGCCAAGAAATACAACAGAAAGTCTGTCACAGCTGTGCATAAGGCAAATATCATGAAGTTAGGTGACGGTCTGTTCAGAAATATAATAACTGAAATTGGCCAAAAAGAATATCCTGATATTGACGTATCGTCCATCATTGTCGACAATGCCTCCATGCAGGCGGTGGCCAAACCTCATCAATTTGATGTCCTAGTTACCCCTTCAATGTACGGTACCATCTTAGGCAACATTGGCGCTGCTTTGATCGGTGGTCCAGGATTGGTGGCAGGTGCCAACTTTGGCAGGGACTATGCTGTCTTCGAACCAGGTTCCAGACATGTTGGTTTAGATATTAAAGGCCAAAATGTGGCTAACCCAACTGCCATGATCCTTTCCTCCACGTTAATGTTGAACCATTTGGGTTTGAATGAATATGCTACTAGAATCTCAAAGGCAGTTCATGAAACGATCGCAGAAGGTAAGCATACCACTAGAGATATTGGTGGTTCCTCTTCTACTACTGACTTCACGAATGAAATCATCAACAAATTATCTACCATGTAAencoded by YNL037c is deleted using a quadruple gene targeting substrateaccording to the following procedure:

A target sequence upstream of IDH1 gene is amplified from genomic DNA byPCR using the primers IDH1-up-fw and IDH1-up-ry and is fused to the twothirds of the K. lactis URA3 gene to the 5′ end by PCR. Furthermore atarget sequence corresponding to the downstream region of IDH1 isamplified from genomic DNA using the primers IDH1-D-fw and IDH1-d-rv.The downstream target sequence is fused to the two thirds of the K.lactis URA3 gene to the 3′ end by PCR.

The yeast strain FS01528 (MATa ura3 his3) is transformed with the twolinear fusion substrates described above containing the upstream targetregion and the downstream target region of the gene to be deleted fusedto either two thirds of the K. lactis URA3 gene. Transformants areselected on medium lacking uracil and are streak-purified on the samemedium. Transformants are transferred to plates containing 5-FOA.Pop-out recombinants are streak-purified on 5-FOA-containing medium. Theresulting strain has the genotype (MATa ura3 his3 IDH1Δ). Correctdeletion of the IDH1 gene is verified by PCR using the primersIDH1-up-fw and IDH1-D-rv.

Example 40 Mating of Cells, Sporulation, Tetrad Dissection and TetradScoring (Analysis)

Methods for combining genetic features by crossing of strains used inthe examples are well known and are, e.g., described in: Adams, A.,Gottschling, D. E., Kaiser, C. A., and Stearns, T. Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1997). Typically,strains of opposite mating types were allowed to mate, diploids wereselected and transferred to sporulation medium (20 g/l potassiumacetate, 1 g/l glucose, 2.5 g/l yeast extract, pH 7.0) and were allowedto sporulate at 30° C. for approximately 3 days. The asci were dissectedon a YPD plate using a Singer MSM microscope and micromanipulatordissection microscope. The mating types of the resulting tetrads werescored by replica-plating to a lawn of cells with either a or alphamating type, incubating at 30° C. to allow mating, replica-plating tosporulation medium, and visualizing sporulation by illuminating platesunder a 302 nm UV-light source. Auxotrophic markers were scored byreplica plating to drop-out plates. Genetic modifications that could notbe scored by phenotype were scored by colony-PCR. In general, the sameprimer sets that were used for verification of genomic integrations orknockouts were also used for colony-PCR scoring of tetrads.

Example 41 Isolation of Genes Encoding TAL, PAL, C4H, 4CL, and VST1

Tyrosine ammonia lyase (TAL) was isolated from Rhodobacter capsulatus bycodon optimization for expression in S. cerevisiae and was furtherassembled as a synthetic gene as described above.

The isolation of phenylalanine ammonia lyase (PAL2), cinnamate4-hydroxylase (C4H), 4-coumarate:CoenzymeA ligase (4CL1) describedabove.

4-Coumarate:CoenzymeA Ligase (4CL2)

(SEQ ID NO: 60) ATGACGACACAAGATGTGATAGTCAATGATCAGAATGATCAGAAACAGTGTAGTAATGACGTCATTTTCCGATCGAGATTGCCTGATATATACATCCCTAACCACCTCCCACTCCACGACTACATCTTCGAAAATATCTCAGAGTTCGCCGCTAAGCCATGCTTGATCAACGGTCCCACCGGCGAAGTATACACCTACGCCGATGTCCACGTAACATCTCGGAAACTCGCCGCCGGTCTTCATAACCTCGGCGTGAAGCAACACGACGTTGTAATGATCCTCCTCCCGAACTCTCCTGAAGTAGTCCTCACTTTCCTTGCCGCCTCCTTCATCGGCGCAATCACCACCTCCGCGAACCCGTTCTTCACTCCGGCGGAGATTTCTAAACAAGCCAAAGCCTCCGCGGCGAAACTCATCGTCACTCAATCCCGTTACGTCGATAAAATCAAGAACCTCCAAAACGACGGCGTTTTGATCGTCACCACCGACTCCGACGCCATCCCCGAAAACTGCCTCCGTTTCTCCGAGTTAACTCAGTCCGAAGAACCACGAGTGGACTCAATACCGGAGAAGATTTCGCCAGAAGACGTCGTGGCGCTTCCTTTCTCATCCGGCACGACGGGTCTCCCCAAAGGAGTGATGCTAACACACAAAGGTCTAGTCACGAGCGTGGCGCAGCAAGTCGACGGCGAGAATCCGAATCTTTACTTCAACAGAGACGACGTGATCCTCTGTGTCTTGCCTATGTTCCATATATACGCTCTCAACTCCATCATGCTCTGTAGTCTCAGAGTTGGTGCCACGATCTTGATAATGCCTAAGTTCGAAATCACTCTCTTGTTAGAGCAGATACAAAGGTGTAAAGTCACGGTGGCTATGGTCGTGCCACCGATCGTTTTAGCTATCGCGAAGTCGCCGGAGACGGAGAAGTATGATCTGAGCTCGGTTAGGATGGTTAAGTCTGGAGCAGCTCCTCTTGGTAAGGAGCTTGAAGATGCTATTAGTGCTAAGTTTCCTAACGCCAAGCTTGGTCAGGGCTATGGGATGACAGAAGCAGGTCCGGTGCTAGCAATGTCGTTAGGGTTTGCTAAAGAGCCGTTTCCAGTGAAGTCAGGAGCATGTGGTACGGTGGTGAGGAACGCCGAGATGAAGATACTTGATCCAGACACAGGAGATTCTTTGCCTAGGAACAAACCCGGCGAAATATGCATCCGTGGCAACCAAATCATGAAAGGCTATCTCAATGACCCCTTGGCCACGGCATCGACGATCGATAAAGATGGTTGGCTTCACACTGGAGACGTCGGATTTATCGATGATGACGACGAGCTTTTCATTGTGGATAGATTGAAAGAACTCATCAAGTACAAAGGATTTCAAGTGGCTCCAGCTGAGCTAGAGTCTCTCCTCATAGGTCATCCAGAAATCAATGATGTTGCTGTCGTCGCCATGAAGGAAGAAGATGCTGGTGAGGTTCCTGTTGCGTTTGTGGTGAGATCGAAAGATTCAAATATATCCGAAGATGAAATCAAGCAATTCGTGTCAAAACAGGTTGTGTTTTATAAGAGAATCAACAAAGTGTTCTTCACTGACTCTATTCCTAAAGCTCCATCAGGGAAGATATTGAGGAAGGATCTAAGAGCAAGACTA GCAAATGGATTAATGAACTAGwas isolated via PCR from A. thaliana cDNA (BioCat, Heidelberg, Germany)using the forward primer 5′-GCGAATTCTTATGACGACA CAAGATGTGATAGTCAATGAT-3′containing an EcoR1 restriction site and reverse primer5′-GCACTAGTATCCTAGTTCATTAATCCATTT GCTAGTCTTGCT-3′ containing a Spe1restriction site.

The VST1 gene encoding Vitis vinifera (grapevine) resveratrol synthase(Hain et al, 1993) was synthesized by GenScript Corporation (Piscataway,N.J.). The amino acid sequence:

(SEQ ID NO: 63)   1MASVEEFRNA QRAKGPATIL AIGTATPDHC VYQSDYADYY FRVTKSEHMT  51ELKKKFNRIC DKSMIKKRYI HLTEEMLEEH PNIGAYMAPS LNIRQEIITA 101EVPRLGRDAA LKALKEWGQP KSKITHLVFC TTSGVEMPGA DYKLANLLGL 151ETSVRRVMLY HQGCYAGGTV LRTAKDLAEN NAGARVLVVC SEITVVTFRG 201PSEDALDSLV GQALFGDGSS AVIVGSDPDV SIERPLFQLV SAAQTFIPNS 251AGAIAGNLRE VGLTFHLWPN VPTLISENIE KCLTQAFDPL GISDWNSLFW 301IAHPGGPAIL DAVEAKLNLE KKKLEATRHV LSEYGNMSSA CVLFILDEMR 351KKSLKGEKAT TGEGLDWGVL FGFGPGLTIE TVVLHSVPTV TN**was used as template to generate a synthetic gene optimized forexpression in S. cerevisiae:

ATGGCATCCGTAGAGGAGTTCAGAAATGCACAGAGGGCAAAAGGTCCAGCAACCATATTGGCTATTGGAACAGCCACCCCTGATCACTGTGTTTATCAATCTGATTACGCTGATTACTATTTCAGAGTAACTAAAAGTGAACATATGACAGAACTTAAGAAAAAGTTTAATAGAATTTGTGATAAATCTATGATAAAGAAAAGATACATACATCTAACTGAAGAAATGTTAGAGGAACATCCAAATATAGGTGCATATATGGCACCATCTTTGAATATTAGACAAGAAATCATAACAGCCGAGGTACCTAGACTAGGTAGAGACGCAGCCTTGAAAGCTTTAAAGGAATGGGGACAACCAAAATCTAAGATTACACATTTGGTTTTCTGTACAACTTCCGGTGTCGAAATGCCAGGTGCTGATTATAAACTAGCAAACCTATTGGGATTAGAGACCTCTGTTAGAAGAGTTATGTTGTATCATCAAGGTTGTTACGCCGGAGGTACAGTGCTTAGAACTGCTAAGGATTTGGCAGAAAATAACGCCGGTGCTAGGGTTTTAGTCGTCTGCAGTGAAATCACTGTCGTAACTTTCAGAGGTCCATCAGAAGATGCTCTAGACAGTTTGGTCGGACAAGCATTGTTTGGCGATGGATCTTCCGCCGTAATTGTAGGCAGCGATCCTGATGTGTCCATTGAAAGACCACTATTTCAATTAGTTTCTGCTGCTCAAACTTTTATTCCAAATTCCGCCGGTGCCATAGCAGGAAACTTGAGAGAAGTTGGTTTGACTTTTCATTTGTGGCCTAATGTCCCAACCTTAATTTCAGAAAACATCGAAAAATGCTTAACTCAAGCCTTTGACCCATTGGGCATAAGCGACTGGAACTCATTGTTTTGGATTGCTCATCCAGGTGGTCCAGCAATTTTAGACGCAGTGGAGGCAAAACTAAACTTAGAGAAGAAAAAGTTGGAAGCTACAAGACACGTTCTATCAGAGTATGGCAACATGAGCTCTGCCTGCGTTTTATTCATTCTAGATGAGATGAGGAAGAAGTCTTTAAAGGGTGAAAAAGCCACAACCGGAGAAGGTTTAGATTGGGGTGTTCTATTTGGTTTCGGTCCTGGCTTAACAATTGAGACAGTGGTGTTACACTCTGTTCCAACTGTCACTAACTAATGA

(SEQ ID NO: 64). The synthetic VST1 gene was delivered inserted in E.coli pUC57 vector flanked by BamH1 and Xho1 restriction sites. Thesynthetic gene was purified from the pUC57 vector by BamH1/Xho1restriction and purified from agarose gel using the QiaQuick GelExtraction Kit (Qiagen).

Example 42 Construction of a Yeast Vector for Expression of TAL

Plasmid, pESC-URA-TAL, containing the gene encoding tyrosine ammonialyase, TAL, under the control of the divergent GAL1/GAL10 promoter wasconstructed as described above for PAL.

Example 43 Construction of a Yeast Vector for Expression of 4CL

The gene encoding 4CL1 and 4CL2 were isolated as described above. Theamplified 4CL1 PCR-product was digested with Xba1/BamH1 and ligated intoSpe1/BglII digested pESC-TRP vector (Stratagene), resulting in vectorpESC-TRP-4CL. The amplified 4CL2 PCR-product was digested withEcoR1/Spe1 and ligated into EcoR1/Spe1 digested pESC-HIS vector(Stratagene), resulting in vector pESC-HIS-4CL2.

Two different clones of pESC-TRP-4CL1 and pESC-HIS-4CL2 were sequencedto verify the sequence of the cloned gene.

Example 44 Construction of a Yeast Vectors for Expression of 4CL and VST

The gene encoding VST from Vitis vinifera (grape) was isolated asdescribed above. The purified BamH1/Xho1 digested VST gene fragment wasligated into BamH1/Xho1 digested pESC-HIS-4CL2 plasmid or pESC-trp-4CL1plasmid (example 15). The resulting plasmids, pESC-HIS-4CL2-VST andpESC-TRP-4CL1-VST contained the genes encoding 4CL1, 4CL2 and VST underthe control of the divergent GAL1/GAL10 promoter. The sequence of thegene encoding VST was verified by sequencing of two different clones ofpESC-HIS-4CL2-VST and pESC-TRP-4CL1-VST.

Example 45 Expression of the PAL-Pathway to Resveratrol in the Yeast S.cerevisiae Using PAL, C4H, 4CL and VST

Yeast strains containing the appropriate genetic markers weretransformed with the vectors described in examples 36 and 38. Thetransformation of the yeast cell was conducted in accordance withmethods known in the art, for instance, by using competent cells or byelectroporation (see, e.g., Sambrook et al., 1989).

S. cerevisiae strain FS01267 (MATa ura3 trp1) was co-transformed withthe vectors pESC-URA-PAL-C4H and pESC-TRP-4CL1-VST, resulting in thestrain FSSC-PALC4H4CL1VST.

S. cerevisiae strain FS01528 (MATa ura3 his3) was co-transformed withthe vectors pESC-URA-PAL-C4H and pESC-HIS-4CL2-VST, resulting in thestrain FSSC-PALC4H4CL2VST.

Transformants were selected on medium lacking uracil and tryptophan oruracil and histidine and streak purified on the same medium.

Example 46 Expression of the TAL-Pathway to Resveratrol in S. cerevisiaeUsing TAL, 4CL and VST

S. cerevisiae strain FS01528 (MATa ura3 his3) was co-transformed withpESC-URA-TAL (example 42) and pESC-HIS-4CL2-VST (example 44), and thetransformed strain was named FSSC-TAL4CL2VST. Transformants wereselected on medium lacking uracil and histidine and streak purified onthe same medium.

Example 47 Expression of the PAL-Pathway to Resveratrol in S. cerevisiaeStrain Overexpressing Native S. cerevisiae NADP-Cytochrome P450Reductase

FSpADH1-CPR (Mata ura3 his3 pADH1-CPR1) (Example 36) was co-transformedwith the vectors pESC-URA-PAL-C4H and pESC-HIS-4CL2-VST, resulting inthe strain FSSC-PALC4H4CL2VST-pADH1CPR1 (Mata ura3 his3 pADH1-CPR1,pESC-URA-PAL-C4H, pESC-HIS-4CL2-VST).

Example 48 Expression of the PAL-Pathway to Resveratrol in S. cerevisiaeStrain Overexpressing Native S. cerevisiae ACC1 Gene

FS01392 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1 pADH1-FAS2) (example 37)was co-transformed with the vectors pESC-URA-PAL-C4H andpESC-TRP-4CL1-VST, resulting in the strain FS01392-PAL.

As a control the strain FS01372 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1pADH1-FAS2)(Example 37) was also co-transformed with the vectorspESC-URA-PAL-C4H and pESC-TRP-4CL1-VST, resulting in the strainFS01372-PALcon.

Example 49 Expression of the TAL-Pathway to Resveratrol in S. cerevisiaeStrain Overexpressing Native S. cerevisiae ACC1 Gene

FS01392 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1 pADH1-FAS2) (example 37)was co-transformed with the vectors pESC-URA-TAL and pESC-TRP-4CL1-VST,resulting in the strain FS01392-TAL.

As a control the strain FS01372 (MATa ura3 trp1 pTPI1-ACC1 PADH1-FAS1pADH1-FAS2)(Example 37) was also co-transformed with the vectorspESC-TAL and pESC-TRP-4CL1-VST, resulting in the strain FS01372-TALcon.

Example 50 HPLC Analysis of Hydroxyl Stilbenes

For quantitative analysis of cinnamic acid, coumaric acid, pinosylvinand resveratrol, cell free supernatant samples were subjected toseparation by high-performance liquid chromatography (HPLC) AgilentSeries 1100 system (Hewlett Packard) prior to uv-diode-array detectionat λ=306 nm. A Phenomenex (Torrance, Calif., USA) Luna 3 micrometer C18(100×2.00 mm) column was used at 40° C. As mobile phase a gradient ofacetonitrile and milliq water (both containing 50 ppm trifluoroaceticacid) was used at a flow of 0.4 ml/min. The gradient profile was linearfrom 15% acetonitrile to 100% acetonitrile over 20 min. The elutiontimes were approximately 3.4 min. for coumaric acid, 5.5 min. for freetrans-resveratrol and 6.8 min. for cinnamic acid. The elution time wasapproximately 8.8-8.9 minutes for trans-pinosylvin.

Pure pinosylvin standard (>95% pure) was purchased from ArboNova (Turku,Finland). Pure resveratrol standard was purchased from Cayman chemicalcompany, whereas pure coumaric acid and cinnamic acid standards werepurchased from Sigma.

Example 51 Shake Flask Cultivations of Strain Overexpressing CPR

The metabolically engineered recombinant yeast strain with overexpressedCPR, FSSC-PALC4H4CL2VST-pADH1CPR1 (example 19), was inoculated to aninitial optical density of 0.1 and grown in 100 ml defined mineralmedium (Verduyn et al, 1992) that contained vitamins, trace elements, 3g/l glucose and 40 g/l galactose for induction of the PAL-pathway genes.The control strain FSSC-PALC4H4CL2VST (example 17) was inoculated in thesame way in a second shake flask for control comparison.

The 500 ml stoppered shake flasks were incubated for three days at 30°C. and 110 rpm. At 72 hours 1 ml samples were taken out from thecultivations, cells were removed by 1 minute centrifugation (13000 rpm,micro centrifuge), and the cell free supernatant was analyzed with HPLC.

The engineered strain overexpressing CPR produced 12.0 mg/l resveratrolcompared to the control strain that produced 1.0 mg/l resveratrol after72 hours cultivation.

Resveratrol Strain (mg/l) Control 1.0 FSSC-PALC4H4CL2VST OverexpressedCPR 12.0 FSSC-PALC4H4CL2VST-pADH1CPR1

Example 52 Shake Flask Cultivations of Strains Overexpressing ACC1

The metabolically engineered recombinant yeast strains withoverexpressed ACC1, FS01392-PAL (example 20a) and FS01392-TAL (example20b), were inoculated to an initial optical density of 0.1 and grown in100 ml defined mineral medium (Verduyn et al, 1992) that contained,vitamins, trace elements, 3 g/l glucose and 40 g/l galactose forinduction of the PAL-pathway genes. After 24 hours 50 mg coumaric acid(Sigma) precursor dissolved in 1 ml 70% ethanol was added correspondingto a concentration of 500 mg/l coumaric acid in the shake flasks.

The control strains, FS01372-PALcon (example 20a) and FS01372-TAlcon(example 20b), were inoculated and grown in the same way in a secondshake flask for control comparison.

The 500 ml stoppered shake flasks were incubated for three days at 30°C. and 110 rpm. At 68 hours 1 ml samples were taken out from thecultivations, cells were removed by 1 minute centrifugation (13000 rpm,micro centrifuge), and the cell free supernatant was analyzed with HPLC.

The engineered strain FS01392-PAL (overexpressing ACC1 and thePAL-pathway genes produced) 119 mg/l resveratrol and its control strainFS01372-PALcon produced 104 mg/l resveratrol, corresponding to a 14%increase in the engineered strain.

The engineered strain FS01392-TAL (overexpressing ACC1 and theTAL-pathway genes produced) 99.5 mg/l resveratrol and its control strainFS01372-TALcon produced 69 mg/l resveratrol, corresponding to a 44%increase in the engineered strain.

Resveratrol Strain (mg/l)* FS01392-PAL 119.0 Overexpressed ACC1Control-PAL 104.0 FS01372-PALcon FS01392-TAL 99.5 Overexpressed ACC1Control-TAL 69.0 FS01372-TALcon *In these experiments 500 mg/l coumaricacid was added to the shake flasks.

Example 53 Resveratrol Content of Genetically Engineered Yeast Cells

The resveratrol content of yeast cells genetically engineered to produceresveratrol as described in Example 9 was determined. Levels of from0.44 to 0.53 μg/g were found.

Example 53 Determination of Intracellular and Extracellular Levels ofStilbenoids in a Continuous Culture of PALCPR

The yeast strain with overexpressed CPR, FSSC-PALC4H4CL2VST-pADH1CPR1(see Example 47) was grown in a carbon-limited continuous culture with aworking volume of 1 liter. The culture was fed with a defined mediumaccording to Verduyn et al. (1992), containing: 5.0 g/L (NH₄)₂SO₄; 3.0g/L KH₂PO₄; 0.5 g/L MgSO₄.7H20; trace metals and vitamins and 5 g/lglucose and 35 g/l galactose as the grdwth-limiting nutrients. Antifoam(300 μl/L, Sigma A-8436) was added to avoid foaming. The carbon sourcewas autoclaved separately from the mineral medium and afterwards addedto the fermentor. In addition, the vitamin and trace metal solutionswere added to the fermentor by sterile filtration followingautoclavation and cooling of the medium. The fermentor system was fromSartorius BBI systems and consisted of a baffled 3-liter reactor vesselwith 1 liter working volume equipped with Biostat B Plus controller. Thereactor vessel was equipped with two Rushton turbines which wererotating at either 1000 rpm, the temperature was kept at 30±1° C., andthe pH was kept at 5.5±0.2 by automatic addition of 2M KOH. The gasflowwas controlled by a mass flow controller and was set to 1.5 vvm (1.5l/min). The off-gas was led through a cooled condenser, and was analyzedfor O₂ and CO₂ (Model 1308, Innova, Denmark). An initial batch culturewith 35 g/l galactose was started by inoculation of the culture with 10ml of an exponentional growing shakeflask culture containing 5 g/lglucose and 35 g/l galactose. The batch cultivation was switched to acontinuous mode by feeding the same medium continuously to the reactor.The dilution rate was controlled on a constant level basis, aiming atD=0.050 h⁻¹. The continuous culture was regarded to be in steady statewhen both the dilution rate and off-gas signal had not changed for atleast five residence times, and when the metabolite concentrations intwo successive samples taken at intervals of 1 residence time, deviatedby less than 3%. The dissolved-oxygen concentration, which wascontinuously monitored, was kept above 60% of air saturation. Under saidconditions the strain consumed all the galactose, and mainly producedbiomass and CO₂, and only minor amounts of ethanol. Moreover, the RQ wasclose to unity, indicating that metabolism was predominantly inrespirative mode.

For the determination of stilbenoids, samples were taken atapproximately 300 hrs into fermentation corresponding to 15 residencetimes. Cells were harvested by centrifugation 5000 g for 5 minutes. Forthe determination of extracellular levels of stilbenoids, an aliquot of25 ml of supernatant was extracted once with 10 ml ethyl acetate. Theethyl acetate was freeze dried and the dry product redissolved in 0.6 mlmethanol. The samples were than 50-fold diluted in water transferredinto HPLC vials, and analyzed by HPLC. Furthermore, to evaluate whetherthe level of stilbenoids that was produced exceeded the solubility ofthe medium, or were either bound to the cell-membranes 1 ml aliquots ofcell culture, thus including both cells and medium, were mixed with 1 mlof 100% ethanol, and mixed vigorously prior to centrifugation. Thesupernatant was then transferred into HPLC vials and directly analyzedfor the content of stilbenoids. For the determination of intracellularlevels of stilbenoids, an aliquot of 50 ml culture was sampled, andcells and medium were separated by centrifugation. The pellet was washedwith 50 ml of water to remove any stilbenoids that were cell-bound ortrapped into the pellet; after re-centrifugation the pellet was thendissolved in 1 ml water. The resulting cell suspension was distributedinto extraction tubes and broken with glass beads using a fast-prepmachine. The crude extracts were pooled into 10 ml of 100% methanol, andextracted in a rotary chamber for 24 hours in a dark cold room at 4° C.Thereafter, the cell debris was removed via centrifugation for 5 min. at5000 g and the remaining methanol was removed by freeze-dryingovernight. The dry residue was redissolved in 0.4 ml methanol and 0.1 mlwater. The samples were than 50-fold diluted in water and thentransferred into HPLC vials, and analyzed by HPLC.

The Following Table Summarizes the Results:

Intracellular and extracellular levels of stilbenoids of PALCPRcontinuous culture at 300 hrs. 5 g/l glucose, 35 g/l galactose, D =0.05, pH = 5.5., 1000 rpm Resveratrol Pinosylvin Resveratrol PinosylvinResveratrol Pinosylvin Resveratrol Pinosylvin IntracelullarIntracelullar Extracelullar Extracelullar Extracellular ExtracellularTotal Total (a) (b) (c) (d) In EtOH (e) In EtOH (f) (a + e) (b + f) 2.2716.45 23.69 12.55 23.65 113.57  25.92 130.02 8.74 12.65 91.43  9.6591.26  87.35 100.00 100.00 0.25  1.83 — — — — — —

Intracellular levels of stilbenoids were expressed in mg per grambiomass (dry weight), according to the calculation explained in thefollowing section. The concentration of resveratrol and pinosylvin inthe extract was determined as 227 mg/l and 1646 mg/l respectively; thevolume of the extract was 0.5 ml, hence the absolute amount ofresveratrol and pinosylvin extracted was 0.5*227/1000=0.1135 mg and0.5*1646/1000=0.8230 mg respectively. The stilbenoids were extractedfrom a 50 ml culture-aliquot and hence the intracellular concentrationsof resveratrol and pinosylvin expressed per liter culture were0.1135*(1000/50)=2.27 mg/l and 0.8230*(1000/50)=16.46 mg/l. The biomassconcentration of said culture was 9 g/l. The intracellular resveratrol-and pinosylvin levels expressed per gram dry weight therefore were2.27/9=0.25 mg/g dry weight and 16.46/9=1.83 mg/g dry weightrespectively.

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The following is a summary of the nucleotide and amino acid sequencesappearing herein:

SEQ ID NO: 1 is a nucleotide sequence from Arabidopsis thaliana encodinga phenylalanine ammonia lyase (PAL2).SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.SEQ ID NO: 3 is a nucleotide sequence from Arabidopsis thaliana encodinga cinnamate 4-hydroxylase (C4H).SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.SEQ ID NO: 5 is a nucleotide sequence from Arabidopsis thaliana encodinga 4-coumarate:CoenzymeA ligase (4CL1).SEQ ID NO: 6 is the amino acid sequence encoded by SEQ ID NO: 5.SEQ ID NO: 7 is a nucleotide sequence from Rheum tataricum encoding aresveratrol synthase (VST).SEQ ID NO: 8 is the amino acid sequence encoded by SEQ ID NO: 7.SEQ ID NO: 9 is a nucleotide sequence from Rheum tataricum encoding aresveratrol synthase (VST), which is codon-optimized for expression inS. cerevisiae.SEQ ID NO: 10 is the amino acid sequence encoded by SEQ ID NO: 9.SEQ ID NO: 11 is a nucleotide sequence from Rhodobacter capsulatusencoding a tyrosine ammonia lyase (TAL).SEQ ID NO: 12 is the amino acid sequence encoded by SEQ ID NO: 11.SEQ ID NO: 13 is a nucleotide sequence from Rhodobacter capsulatusencoding a tyrosine ammonia lyase (TAL), which is codon-optimized forexpression in S. cerevisiae.SEQ ID NO: 14 is the amino acid sequence encoded by SEQ ID NO: 13.SEQ ID NO: 15 is a nucleotide sequence from S. cerevisiae encoding aNADPH:cytochrome P450 reductase (CPR1).SEQ ID NO: 16 is the amino acid sequence encoded by SEQ ID NO: 15.SEQ ID NO: 17 is a nucleotide sequence from Arabidopsis thalianusencoding a NADPH:cytochrome P450 reductase (AR2).SEQ ID NO: 18 is the amino acid sequence encoded by SEQ ID NO: 17.SEQ ID NOs 19-32 are primer sequences appearing in Table 1, Example 1.SEQ ID NOs 33-34 are primer sequences appearing in Example 16.SEQ ID NOs 35-38 are primer sequences appearing in Example 17.SEQ ID NOs 39-46 are primer sequences appearing in Example 36, Table 1.SEQ ID NOs 47-58 are primer sequences appearing in Example 37, Table 2.SEQ ID NO: 59 is the gene sequence appearing in Example 39.SEQ ID NO: 60 is the first gene sequence appearing in Example 41.SEQ ID NOs 61-62 are primer sequences appearing in Example 41.SEQ ID NO: 63 is the VST1 amino acid sequence in Example 41.SEQ ID NO. 64 is the second gene sequence appearing in Example 41.

1. A genetically engineered micro-organism comprising an engineeredoperative metabolic pathway producing resveratrol, or an oligomeric orglycosidically-bound derivative thereof, wherein the engineeredoperative metabolic pathway produces: a) 4-coumaric acid fromL-phenylalanine catalysed by a phenylalanine ammonia lyase and acinnamate 4-hydroxylase expressed in the micro-organism or from tyrosinecatalysed by a phenylalanine ammonia lyase or a tyrosine ammonia lyaseexpressed in said micro-organism; and b) 4-coumaroyl-CoA from 4-coumaricacid catalysed by a 4-coumarate-CoA ligase expressed in themicro-organism; wherein the micro-organism further comprises a more thana native expression level of an acetyl coenzymeA carboxylase (ACC1)enzyme.
 2. The micro-organism of claim 1, wherein the resveratrol isproduced in the micro-organism that is cultured in a media with a carbonsubstrate and does not require an external source of phenylalanine,tyrosine, cinnamic acid or coumaric acid, and resveratrol is producedfrom the 4-coumaroyl-CoA by a resveratrol synthase expressed in themicro-organism.
 3. The micro-organism of claim 1, wherein the more thannative expression level of the ACC1 enzyme has been provided byreplacing a native promoter of a gene expressing the ACC1 enzyme with apromoter providing a higher level of expression.
 4. The micro-organismof claim 3, wherein the native promoter is replaced with a strongconstitutive yeast promoter.
 5. The micro-organism of claim 4, whereinthe strong constitutive promoter is a yeast promoter selected from thepromoters for yeast genes: triosephoshosphate dehydrogenase 3 (TDH3),alcohol dehydrogenase 1 (ADH1), triose phosphate isomerase 1 (TPI1) 1actin (ACTT), glyceraldehyde-3-phosphate dehydrogenase (GPD) andphosphoglucose isomerase (PGI).
 6. The micro-organism of claim 1,wherein the more than native expression level of the ACC1 enzyme hasbeen provided by recombinantly introducing into the micro-organism atleast one copy of an exogenous genetic sequence encoding the ACC1enzyme.
 7. The micro-organism of claim 1, wherein the ACC1 enzyme isfrom Saccharomyces cerevisiae.
 8. The micro-organism of claim 1, furthercomprising a more than a native expression level of a NADPH:cytochromeP450 reductase (CPR) enzyme.
 9. The micro-organism of claim 8, whereinthe more than native expression level of the CPR enzyme has beenprovided by replacing a native promoter of a gene expressing the CPRenzyme with a promoter providing a higher level of expression.
 10. Themicro-organism of claim 9, wherein the native promoter is replaced witha strong constitutive yeast promoter.
 11. The micro-organism of claim10, wherein the strong constitutive promoter is a yeast promoterselected from the promoters for yeast genes: triosephoshosphatedehydrogenase 3 (TDH3), alcohol dehydrogenase 1 (ADH1), triose phosphateisomerase 1 (TPI1) 1 actin (ACTT), glyceraldehyde-3-phosphatedehydrogenase (GPD) and phosphoglucose isomerase (PGI).
 12. Themicro-organism of claim 8, wherein the more than native expression levelof the CPR enzyme has been provided by recombinantly introducing intothe micro-organism at least one copy of an exogenous genetic sequenceencoding the CPR enzyme.
 13. The micro-organism of claim 8, wherein theCPR enzyme is from Saccharomyces cerevisiae.
 14. The micro-organism ofclaim 1, wherein the micro-organism is a bacterium belonging to a genusselected from: Bacillus, Escherichia, Lactobacillus, Lactococcus,Corynebacterium, Acetobacter, Acinetobacter, and Pseudomonas.
 15. Themicro-organism of claim 14, wherein the micro-organism is Escherichiacoli.
 16. The micro-organism of claim 1, wherein the micro-organism is ayeast belonging to a genus selected from: Saccharomyces, Kluyveromyces,Pichia, Debaromyces, Hansenula, Pichia, Zygosaccharomyces andSchizosaccharomyces.
 17. The micro-organism of claim 16, wherein themicro-organism is Saccharomyces cerevisiae.
 18. The micro-organism ofclaim 1, wherein the micro-organism is a filamentous fungus belonging toa genus selected from: Rhizopus, Fusidium, Gibberella and Trichoderma.19. The micro-organism of claim 1, wherein the operative metabolicpathway produces at least 0.25 milligrams resveratrol per gram on a dryweight basis of the genetically engineered micro-organism.
 20. A methodfor producing resveratrol or an oligomeric or glycosidically-boundderivative thereof, comprising: a) cultivating the geneticallyengineered micro-organism of claim 1; and b) recovering the resveratrol,or the oligomeric or glycosidically-bound derivative thereof, from theculture media.